Method for characterising a double stranded nucleic acid using a nano-pore and anchor molecules at both ends of said nucleic acid

ABSTRACT

The invention relates to a method for method of characterising, such as sequencing, a target double stranded polynucleotide. The polynucleotide is coupled to a membrane using at least two adaptors with different strengths of coupling to the membrane.

FIELD OF THE INVENTION

The invention relates to a method of characterising, such as sequencing,a target double stranded polynucleotide. The polynucleotide is coupledto a membrane using at least two adaptors with different strengths ofcoupling to the membrane.

BACKGROUND OF THE INVENTION

There is currently a need for rapid and cheap polynucleotide (e.g. DNAor RNA) sequencing and identification technologies across a wide rangeof applications. Existing technologies are slow and expensive mainlybecause they rely on amplification techniques to produce large volumesof polynucleotide and require a high quantity of specialist fluorescentchemicals for signal detection.

Transmembrane pores (nanopores) have great potential as direct,electrical biosensors for polymers and a variety of small molecules. Inparticular, recent focus has been given to nanopores as a potential DNAsequencing technology.

When a potential is applied across a nanopore, there is a change in thecurrent flow when an analyte, such as a nucleotide, resides transientlyin the barrel for a certain period of time. Nanopore detection of thenucleotide gives a current change of known signature and duration. Inthe strand sequencing method, a single polynucleotide strand is passedthrough the pore and the identities of the nucleotides are derived.Strand sequencing can involve the use of a polynucleotide bindingprotein to control the movement of the polynucleotide through the pore.

It has previously been demonstrated that ultra low concentration analytedelivery can be achieved by coupling the analyte to a membrane in whichthe relevant detector is present. This lowers by several orders ofmagnitude the amount of analyte required in order to be detected (WO2012/164270).

It has also been shown that double stranded polynucleotides can beeffectively characterised using strand sequencing if they are modifiedto include a Y adaptor (a double stranded stem and two non-complementaryarms) containing a leader sequence and a hairpin loop adaptor (WO2013/014451). It is preferred that that Y adaptor containing the leadersequence is attached to one end of the polynucleotide and the hairpinloop adaptor is attached to the other end. The leader sequencepreferentially threads into the nanopore and the hairpin loop connectingthe two strands of the polynucleotide allows both strands to beinvestigated as the polynucleotide unzips and moves through the pore.This is advantageous because it doubles the amount of informationobtained from a single double stranded polynucleotide. Moreover, becausethe sequences in the two strands are complementary, the information fromthe two strands can be combined informatically. This mechanism providesan orthogonal proof-reading capability that provides higher confidenceobservations. When Y adaptors and hairpin loops are used together inthis way, the Y adaptor typically contains an anchor which couples thepolynucleotide to the membrane containing the nanopore. In someinstances, double stranded polynucleotides having Y adaptors at bothends are produced in the sample preparation. The presence of the leadersequence and the anchor in the Y adaptors means that the system istypically biased towards characterising these polynucleotides. However,the lack of the hairpin loop linking the two strands in suchpolynucleotides means that only one strand is investigated.

SUMMARY OF THE INVENTION

The inventors have surprisingly demonstrated that it is possible toavoid a bias towards double stranded polynucleotides comprising a Yadaptor at both ends by including two anchors in the system. Theinventors have used a Y adaptor comprising one or more first anchors forcoupling the polynucleotide to the membrane and a hairpin loop adaptorcomprising one or more second anchors for coupling the polynucleotide tothe membrane. The hairpin loop adaptor couples the polynucleotide to themembrane with more strength than the Y adaptor.

Accordingly, the invention provides a method of characterising a targetdouble stranded polynucleotide using a transmembrane pore in a membrane,comprising:

a) providing the target double stranded polynucleotide with a Y adaptorat one end and a hairpin loop adaptor at the other end, wherein the Yadaptor comprises one or more first anchors for coupling thepolynucleotide to the membrane, wherein the hairpin loop adaptorcomprises one or more second anchors for coupling the polynucleotide tothe membrane and wherein the strength of coupling of the hairpin loopadaptor to the membrane is greater than the strength of coupling of theY adaptor to the membrane;

b) contacting the polynucleotide provided in step a) with thetransmembrane pore such that at least one strand of the polynucleotidemoves through the pore; and

c) taking one or more measurements as the at least one strand of thepolynucleotide moves with respect to the pore wherein the measurementsare indicative of one or more characteristics of the at least one strandof the polynucleotide and thereby characterising the double strandedtarget polynucleotide.

The invention also provides:

a method for modifying a target double stranded polynucleotide forcharacterisation using a transmembrane pore in a membrane, comprisingligating a Y adaptor to one end of the polynucleotide and ligating ahairpin loop adaptor to the other end of the polynucleotide, wherein theY adaptor comprises one or more first anchors for coupling thepolynucleotide to the membrane, wherein the hairpin loop adaptorcomprises one or more second anchors for coupling the polynucleotide tothe membrane and wherein the strength of coupling of the hairpin loopadaptor to the membrane is greater than the strength of coupling of theY adaptor to the membrane, and thereby providing a modified targetdouble stranded polynucleotide;

a method for modifying a target double stranded polynucleotide forcharacterisation using a transmembrane pore in a membrane, comprising:(a) ligating a Y adaptor to one end of the polynucleotide and ligating ahairpin loop adaptor to the other end of the polynucleotide; and (b)attaching to the Y adaptor one or more first anchors for coupling thepolynucleotide to the membrane, attaching to the hairpin loop adaptorone or more second anchors for coupling the polynucleotide to themembrane and thereby providing a modified target double strandedpolynucleotide; wherein the strength of coupling of the hairpin loopadaptor to the membrane is greater than the strength of coupling of theY adaptor to the membrane;

a target double stranded polynucleotide modified using the method of theinvention;

a method for modifying a target double stranded polynucleotide forcharacterisation using a transmembrane pore in a membrane, comprisingcontacting the target polynucleotide with a MuA transposase and apopulation of double stranded MuA substrates, wherein a proportion ofthe substrates in the population are Y adaptors comprising one or morefirst anchors for coupling the polynucleotide to the membrane, wherein aproportion of the substrates in the population are hairpin loop adaptorscomprising one or more second anchors for coupling the polynucleotide tothe membrane and wherein the strength of coupling of the hairpin loopadaptor to the membrane is greater than the strength of coupling of theY adaptor to the membrane, and thereby producing a plurality of modifieddouble stranded polynucleotides;

a method for modifying a target double stranded polynucleotide forcharacterisation using a transmembrane pore in a membrane, comprising:(a) contacting the target polynucleotide with a MuA transposase and apopulation of double stranded MuA substrates, wherein a proportion ofthe substrates in the population are Y adaptors and wherein a proportionof the substrates in the population are hairpin loop adaptors, such thatthe transposase fragments the target polynucleotide and ligates asubstrate to one or both ends of the double stranded fragments andthereby produces a plurality of fragment/substrate constructs; and (b)attaching to the Y adaptors in the plurality of fragment/substrateconstructs one or more first anchors and attaching to the hairpin loopadaptors in the plurality of fragment/substrate constructs one or moresecond anchors and thereby producing a plurality of modified doublestranded polynucleotides; wherein the strength of coupling of eachhairpin loop adaptor to the membrane is greater than the strength ofcoupling of each Y adaptor to the membrane;

a plurality of polynucleotides modified using the method of theinvention;

a method of characterising a target double stranded polynucleotide usinga transmembrane pore in a membrane, comprising:

a) providing the target double stranded polynucleotide with a Y adaptorat one end and a hairpin loop adaptor at the other end, wherein the Yadaptor comprises one or more first anchors for coupling thepolynucleotide to the membrane, wherein the hairpin loop adaptorcomprises one or more second anchors for coupling the polynucleotide tothe membrane and wherein the strength of coupling of the hairpin loopadaptor to the membrane is greater than the strength of coupling of theY adaptor to the membrane;

b) contacting the polynucleotide provided in step a) with thetransmembrane pore, a polymerase and labelled nucleotides such thatphosphate labelled species are sequentially released when nucleotidesare added to the polynucleotide by the polymerase, wherein the phosphatespecies contain a label specific for each nucleotide; and

c) detecting the phosphate labelled species using the pore and therebycharacterising the double stranded target polynucleotide;

a pair of adaptors for modifying a target double stranded polynucleotidefor characterisation using a transmembrane pore in a membrane, whereinone adaptor is a Y adaptor comprising one or more first anchors forcoupling the polynucleotide to the membrane, wherein the other adaptoris a hairpin loop adaptor comprising one or more second anchors forcoupling the polynucleotide to the membrane and wherein the strength ofcoupling of the hairpin loop adaptor to the membrane is greater than thestrength of coupling of the Y adaptor to the membrane;

a population of adaptors for modifying a target polynucleotide forcharacterisation using a transmembrane pore in a membrane, wherein aproportion of the adaptors are Y adaptors comprising one or more firstanchors for coupling the polynucleotide to the membrane, wherein aproportion of the adaptors are hairpin loop adaptors comprising one ormore second anchors for coupling the polynucleotide to the membrane andwherein the strength of coupling of the hairpin loop adaptors to themembrane is greater than the strength of coupling of the Y adaptors tothe membrane; and

a kit for modifying a target polynucleotide comprising (a) a pair ofadaptors or a population of adaptors of the invention and (b) a MuAtransposase.

DESCRIPTION OF THE FIGURES

FIG. 1 shows in sections (A) and (B) cartoon representations of the DNAconstructs used in Example 2—four iSpC3 spacers are shown as a cross andfour 5-nitroindoles as a black 0, the cholesterol coupling agent as agrey oval and the palmitate coupling agent as a grey square; labelsal-10 are described in full in Example 1.

FIG. 2 shows in sections (A) and (B) cartoon representations of the DNAconstructs used in Example 3—four iSpC3 spacers are shown as a cross andfour 5-nitroindoles as a black 0, the cholesterol coupling agent as agrey oval and the palmitate anchor as a grey square; labels al-9 aredescribed in full in Example 1.

FIG. 3 shows the proportion of helicase-controlled DNA movementsdetected for construct 1 where strand a2 translocated through thenanopore and construct 2 where strand a2 translocated through thenanopore. The x-axis shows the position along the lambda DNA sequence towhich that the helicase-controlled DNA movements mapped (they were allconcentrated in the same region as strands a1 and a2 corresponded to thetemplate and template complement of the region of lambda between 45,042bp and 48,487 bp). The Y-axis shows the proportion of strand a1(construct 1) helicase-controlled DNA movements (labelled a1) to stranda2 (construct 2) helicase controlled DNA movements (labelled a2). Thiswas observed to be approximately 50:50 when a single cholesterol wasused to couple the constructs to the membrane. This control showed thatif the same coupling agent was used to couple different constructs tothe membrane, the number of helicase controlled DNA movements would havebeen approximately equal for each construct e.g. no bias was observed.

FIG. 4 shows the proportion of helicase-controlled DNA movementsdetected for construct 2 where strand a2 translocated through thenanopore and construct 3 where strand a1 translocated through thenanopore. The x-axis shows the position along the lambda DNA sequence towhich that the helicase-controlled DNA movements mapped (they were allconcentrated in the same region as strands a1 and a2 corresponded to thetemplate and template complement of the region of lambda between 45,042bp and 48,487 bp). The Y-axis shows the proportion of strand a2(construct 2) helicase-controlled DNA movements (labelled a2) to stranda1 (construct 3) helicase controlled DNA movements (labelled a1). Thiswas observed to be approximately 5:95 when a single cholesterol was usedto couple construct 2 to the membrane in comparison to when acholesterol and a palmitate were used to couple construct 3 to themembrane. This experiment showed that when two coupling agents were usedto couple a construct to the membrane (where one was a stronger couplingagent than the other e.g. cholesterol was stronger than palmitate) incomparison to a single coupling agent (cholesterol) the number ofhelicase-controlled DNA movements was strongly biased towards the doublycoupled construct over the singly coupled construct.

FIG. 5 shows the proportion of helicase-controlled DNA movementsdetected for construct 4 where strand a2 translocated through thenanopore and construct 5 where strand a1 or a2 translocated through thenanopore. The x-axis shows the position along the lambda DNA sequence towhich that the helicase-controlled DNA movements mapped (they were allconcentrated in the same region as strands a1 and a2 corresponded to thetemplate and template complement of the region of lambda between 45,042bp and 48,487 bp). The Y-axis shows the proportion of strand a1(construct 4 or 5) helicase-controlled DNA movements (labelled a1) tostrand a2 (construct 5 only) helicase controlled DNA movements (labelleda2). This was observed to be approximately 33:66 (a2:a1 helicasecontrolled DNA movements) when two cholesterols were used to couple bothconstructs to the membrane. The 2:1 bias towards a1 helicase controlledDNA movements was predicted because a2 movements would only be detectedif construct 4 was captured by the nanopore, whereas a1 movements wouldbe detected from construct 4 and 5, therefore, twice as many a1movements were expected.

FIG. 6 shows the proportion of helicase-controlled DNA movementsdetected for construct 3 where strand a2 translocated through thenanopore and construct 6 where strand a1 or a2 translocated through thenanopore. The x-axis shows the position along the lambda DNA sequence towhich that the helicase-controlled DNA movements mapped (they were allconcentrated in the same region as strands a 1 and a2 corresponded tothe template and template complement of the region of lambda between45,042 bp and 48,487 bp). The Y-axis shows the proportion of strand a1(construct 3 or 6) helicase-controlled DNA movements (labelled a1) tostrand a2 (construct 6 only) helicase controlled DNA movements (labelleda2). This was observed to be approximately 5:95 (a2:a1 helicasecontrolled DNA movements) when the combination of a cholesterol and apalmitate were used to couple construct 3 to the membrane and twopalmitates were used to couple construct 6 to the membrane. The biastowards a1 helicase controlled DNA movements in comparison to thecontrol experiment shown in FIG. 5 illustrated that by using thecombination of two different coupling agents of differing strength itwas possible to select for this construct over the construct whichcontained two coupling agents of similar strength.

FIG. 7 shows in sections (A) and (B) cartoon representations of the DNAconstructs used in Example 4—four iSpC3 spacers are shown as a cross andfour 5-nitroindoles as a black 0, the cholesterol coupling agent as agrey oval and the alternative coupling agents investigated in thisexample as a black triangle; labels a1-10(a-c) are described in full inExample 1 and 4.

FIG. 8 shows the proportion of helicase-controlled DNA movementsdetected for construct 1 (coupled to the membrane using a singlecholesterol) where strand a 1 translocated through the nanopore andconstruct 2 (also coupled to the membrane using a single cholesterol)where strand a2 translocated through the nanopore. The x-axis shows theposition along the lambda DNA sequence to which that thehelicase-controlled DNA movements mapped (they were all concentrated inthe same region as strands a1 and a2 corresponded to the template andtemplate complement of the region of lambda between 45,042 bp and 48,487bp). The Y-axis shows the proportion of strand a1 (construct 1)helicase-controlled DNA movements (labelled a1) to strand a2 (construct2) helicase controlled DNA movements (labelled a2). This was observed tobe approximately 50:50 when a single cholesterol was used to couple eachconstruct to the membrane. This control showed that if the same couplingagent was used to couple different constructs to the membrane, thenumber of helicase controlled DNA movements would have beenapproximately equal for each construct e.g. no bias was observed.

FIG. 9 shows the proportion of helicase-controlled DNA movementsdetected for construct 1 (coupled to the membrane using a singlecholesterol) where strand a1 translocated through the nanopore andconstruct 7 a (coupled to the membrane using a fragment of DNA which hadtwo cholesterol TEG's attached, each shown as a black triangle) wherestrand a2 translocated through the nanopore. The x-axis shows theposition along the lambda DNA sequence to which that thehelicase-controlled DNA movements mapped (they were all concentrated inthe same region as strands a1 and a2 corresponded to the template andtemplate complement of the region of lambda between 45,042 bp and 48,487bp). The Y-axis shows the proportion of strand a1 (construct 1)helicase-controlled DNA movements (labelled a1) to strand a2 (construct7a) helicase controlled DNA movements (labelled a2). This was observedto be approximately 50:50 when a single cholesterol was used to coupleconstruct 1 to the membrane in comparison to when two cholesterols wereused to couple construct 7a to the membrane. This experiment showed thatwhen two cholesterols attached to the same fragment of DNA were used tocouple construct 7a to the membrane, in comparison to a singlecholesterol, the number of helicase-controlled DNA movements detectedshowed no bias towards either construct. Therefore, the strength ofcoupling observed for two cholesterols in the same DNA fragment wassimilar to a single cholesterol.

FIG. 10 shows the proportion of helicase-controlled DNA movementsdetected for construct 1 (coupled to the membrane using a singlecholesterol) where strand a1 translocated through the nanopore andconstruct 7b (coupled to the membrane using a tocopherol, shown as ablack triangle) where strand a2 translocated through the nanopore. Thex-axis shows the position along the lambda DNA sequence to which thatthe helicase-controlled DNA movements mapped (they were all concentratedin the same region as strands a1 and a2 corresponded to the template andtemplate complement of the region of lambda between 45,042 bp and 48,487bp). The Y-axis shows the proportion of strand a1 (construct 1)helicase-controlled DNA movements (labelled al) to strand a2 (construct7b) helicase controlled DNA movements (labelled a2). This was observedto be approximately 35:65 when a single cholesterol was used to coupleconstruct 1 to the membrane in comparison to the single tocopherol usedto couple construct 7b to the membrane. This experiment showed that whentocopherol was used to couple construct 7b to the membrane in comparisonto a single cholesterol the number of helicase-controlled DNA movementsdetected showed a bias towards the construct coupled using tocopherol.Therefore, the strength of coupling observed for tocopherol was slightlystronger than that observed for cholesterol.

FIG. 11 shows the proportion of helicase-controlled DNA movementsdetected for construct 1 (coupled to the membrane using a singlecholesterol) where strand a1 translocated through the nanopore andconstruct 7c (coupled to the membrane using a palmitate, shown as ablack triangle) where strand a2 translocated through the nanopore. Thex-axis shows the position along the lambda DNA sequence to which thatthe helicase-controlled DNA movements mapped (they were all concentratedin the same region as strands a1 and a2 corresponded to the template andtemplate complement of the region of lambda between 45,042 bp and 48,487bp). The Y-axis shows the proportion of strand a1 (construct 1)helicase-controlled DNA movements (labelled a1) to strand a2 (construct7c) helicase controlled DNA movements (labelled a2). This was observedto be approximately 70:30 when a single cholesterol was used to coupleconstruct 1 to the membrane in comparison to the single palmitate usedto couple construct 7c to the membrane. This experiment showed that whenpalmitate was used to couple construct 7c to the membrane, in comparisonto a single cholesterol, the number of helicase-controlled DNA movementsdetected showed a bias towards the construct coupled using cholesterol.Therefore, the strength of coupling observed for palmitate was weakerthan that observed for cholesterol.

FIG. 12 shows in sections (A) and (B) cartoon representations of the DNAconstructs used in Example 6—twenty-five iSpC3 spacers are representedas three black crosses, four iSp18′s as a grey rectangle, a cholesterolcoupling agent as a grey circle and the palmitate coupling agent as agrey square; labels a11-15 are described in full in Example 5.

FIG. 13 shows the possible constructs produced (as described in Example5) after fragmentation and adapter attachment. Construct 10 has twohairpins adapters and cannot be captured by the nanopore. Construct 8 isthe desired construct which has one Y-adapter and one hairpin, strands Xand Y will both translocate through the nanopore upon capture. Construct11 has two Y-adapters and, therefore, can capture and translocate onlystrand X or strand Y.

FIG. 14 shows the possible constructs produced (as described in Example5) after fragmentation and adapter attachment. Construct 10 has twohairpins adapters and cannot be captured by the nanopore. Construct 9 isthe desired construct which has one Y-adapter and one hairpin, strands Xand Y will both translocate through the nanopore upon capture. Construct12 has two Y-adapters and, therefore, can capture and translocate onlystrand X or strand Y.

FIG. 15 shows in sections (A) and (B) cartoon representations of two ofthe DNA constructs (constructs 13 and 14) used in Example 7—thirty iSpC3spacers are represented as four black crosses, four iSp18′s as a greyrectangle, a cholesterol coupling agent as a grey circle and thepalmitate coupling agent as a grey square; labels a12, a16-21 aredescribed in full in Example 7. Two regions of the DNA construct arelabelled R1 and R2.

FIG. 16 shows in sections (A) and (B) cartoon representations of two ofthe DNA constructs (constructs 15 and 16) used in Example 7—thirty iSpC3spacers are represented as four black crosses, four iSp18′s as a greyrectangle, a cholesterol coupling agent as a grey circle labels a12,a16-a18, a20, a22-23 are described in full in Example 7. Two regions ofthe DNA construct are labelled R1 and R2.

FIG. 17 shows a graph of the % of the helicase-controlled DNA movementsdetected which corresponded to controlled translocation of both regionsR1 and R2 through the nanopore as a proportion of allhelicase-controlled DNA movements observed. The y-axis label=percentage(%) and the x-axis label=DNA construct number (β-16). Each point on thegraph corresponds to a separate experiment (n=3).

FIG. 18 shows a graph of the number of helicase-controlled DNA movementsdetected per nanopore for each of the DNA constructs. The y-axislabel=helicase-controlled DNA movements per nanopore and the x-axislabel=DNA construct number (β-16). Each point on the graph correspondsto a separate experiment (n=3).

DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 shows the codon optimised polynucleotide sequence encodingthe MS-B 1 mutant MspA monomer. This mutant lacks the signal sequenceand includes the following mutations: D90N, D91N, D93N, D118R, D134R andE139K.

SEQ ID NO: 2 shows the amino acid sequence of the mature form of theMS-B 1 mutant of the MspA monomer. This mutant lacks the signal sequenceand includes the following mutations: D90N, D91N, D93N, D118R, D134R andE139K.

SEQ ID NO: 3 shows the polynucleotide sequence encoding one monomer ofα-hemolysin-E111N/K147N (α-HL-NN; Stoddart et al., PNAS, 2009; 106(19):7702-7707).

SEQ ID NO: 4 shows the amino acid sequence of one monomer of α-HL-NN.

SEQ ID NOs: 5 to 7 show the amino acid sequences of MspB, C and D.

SEQ ID NO: 8 shows the polynucleotide sequence encoding the Phi29 DNApolymerase.

SEQ ID NO: 9 shows the amino acid sequence of the Phi29 DNA polymerase.

SEQ ID NO: 10 shows the codon optimised polynucleotide sequence derivedfrom the sbcB gene from E. coli. It encodes the exonuclease I enzyme(EcoExo I) from E. coli.

SEQ ID NO: 11 shows the amino acid sequence of exonuclease I enzyme(EcoExo I) from E. coli.

SEQ ID NO: 12 shows the codon optimised polynucleotide sequence derivedfrom the xthA gene from E. coli. It encodes the exonuclease III enzymefrom E. coli.

SEQ ID NO: 13 shows the amino acid sequence of the exonuclease IIIenzyme from E. coli. This enzyme performs distributive digestion of 5′monophosphate nucleosides from one strand of double stranded DNA (dsDNA)in a 3′-5′ direction. Enzyme initiation on a strand requires a 5′overhang of approximately 4 nucleotides.

SEQ ID NO: 14 shows the codon optimised polynucleotide sequence derivedfrom the recJ gene from T. thermophilus. It encodes the RecJ enzyme fromT. thermophilus (TthRecJ-cd).

SEQ ID NO: 15 shows the amino acid sequence of the RecJ enzyme from T.thermophilus (TthRecJ-cd). This enzyme performs processive digestion of5′ monophosphate nucleosides from ssDNA in a 5′-3′ direction. Enzymeinitiation on a strand requires at least 4 nucleotides.

SEQ ID NO: 16 shows the codon optimised polynucleotide sequence derivedfrom the bacteriophage lambda exo (redX) gene. It encodes thebacteriophage lambda exonuclease.

SEQ ID NO: 17 shows the amino acid sequence of the bacteriophage lambdaexonuclease. The sequence is one of three identical subunits thatassemble into a trimer. The enzyme performs highly processive digestionof nucleotides from one strand of dsDNA, in a 5′-3′ direction(http://www.neb.com/nebecomm/products/productM0262.asp). Enzymeinitiation on a strand preferentially requires a 5′ overhang ofapproximately 4 nucleotides with a 5′ phosphate.

SEQ ID NO: 18 shows the amino acid sequence of He1308 Mbu.

SEQ ID NO: 19 shows the amino acid sequence of He1308 Csy.

SEQ ID NO: 20 shows the amino acid sequence of He1308 Tga.

SEQ ID NO: 21 shows the amino acid sequence of He1308 Mhu.

SEQ ID NO: 22 shows the amino acid sequence of Tral Eco.

SEQ ID NO: 23 shows the amino acid sequence of XPD Mbu.

SEQ ID NO: 24 shows the amino acid sequence of Dda 1993.

SEQ ID NO: 25 shows the amino acid sequence of Trwc Cba.

SEQ ID NO: 26 shows the polynucleotide sequence of the double strandedportion of a MuA substrate of the invention.

SEQ ID NO: 27 shows the polynucleotide sequence of the double strandedportion of a MuA substrate of the invention. This sequence iscomplementary to SEQ ID NO: 26 except that it contains a U at the 3′end.

SEQ ID NO: 28 shows polynucleotide sequence of the overhang strand ofthe double stranded MuA substrate of the invention.

SEQ ID NOs: 29-32 show polynucleotide sequences used in Example 1.

SEQ ID NO: 33-41 show polynucleotide sequences used in Example 1, 2 and3.

SEQ ID NO: 42 shows a polynucleotide sequence used in Example 4.

SEQ ID NO: 43 shows the polynucleotide sequence of the Enterobacteriaphage X. The sequence contains an additional 12 base overhang attachedat the 5′ end of the template strand. The sequence shown here is that ofthe template strand only (the template complement is not shown). Thissequence is used in Example 5.

SEQ ID NO: 44-48 shows the polynucleotide sequences used in Example 5and 6.

SEQ ID NO: 49 shows a polynucleotide sequence used in Example 7. At the5′ end the sequence contains a phosphate group.

SEQ ID NO: 50 shows a polynucleotide sequence used in Example 7. At the3′ end the final thymine in the sequence has a phosphothioate group.

SEQ ID NO: 51 shows a polynucleotide sequence used in Example 7.

SEQ ID NO: 52 shows a polynucleotide sequence used in Example 7.

SEQ ID NO: 53 shows a polynucleotide sequence used in Example 7. At the5′ end the sequence contains a phosphate group.

SEQ ID NO: 54 shows a polynucleotide sequence used in Example 7.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that different applications of the disclosedproducts and methods may be tailored to the specific needs in the art.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments of the invention only, andis not intended to be limiting.

In addition as used in this specification and the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontent clearly dictates otherwise. Thus, for example, reference to “apolynucleotide” includes two or more polynucleotides, reference to “ananchor” refers to two or more anchors, reference to “a helicase”includes two or more helicases, reference to “a transmembrane pore”includes two or more pores and the like.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

Characterisation Method of the Invention

When Y adaptors and hairpin loop adaptors are used together tocharacterise double stranded polynucleotides, the Y adaptor generallycomprises the anchor for coupling the polynucleotide to the membrane anda leader sequence which preferentially threads into the pore. The anchorand more importantly the leader typically result in a bias towards thecharacterisation of double stranded polynucleotides having Y adaptors atboth ends.

When a population of double stranded polynucleotides are modified usingapproximately equal amounts of Y adaptors and hairpin loop adaptors,three distinct groups of modified polynucleotides are produced.Approximately, 50% of the modified polynucleotides have a Y adaptor atone end and a hairpin loop adaptor at the other end (i.e. are “round thecorner” or RTC polynucleotides). It is preferred that these RTCpolynucleotides are characterised using the method disclosed in WO2013/014451. Approximately, 25% of the modified polynucleotides have a Yadaptor at each end (i.e. are double Y polynucleotides) andapproximately 25% of the modified polynucleotides have a hairpin loopadaptor at each end (i.e. are double hairpin polynucleotides). If the Yadaptor comprises the anchor, there is a bias towards characterisationof the double Y polynucleotides because they have two anchors andtherefore couple to the membrane more strongly. If the Y adaptorcomprises the leader, there is a bias towards characterisation of thedouble Y polynucleotides because they have two leaders and thereforepreferentially thread into the pore. The presence of the leadergenerally results in a greater bias than the presence of the anchor. Ifthe Y adaptor comprises both the anchor and the leader, there is thegreatest bias towards characterisation of the double Y polynucleotides.The inventors have seen biases of approximately 50-fold towards double Ypolynucleotides. The double hairpin polynucleotides are not typicallycharacterised because they do not have a single stranded region capableof threading into the nanopore used for characterisation.

The inventors have shown that using one or more anchors on the hairpinloop adaptor not only reduces characterisation of the double Ypolynucleotides, but also reduces the observed throughput of the doubleY polynucleotides significantly. The inventors conclude from this thatthe throughput is dependent on the presence of an anchor at the end ofthe modified polynucleotide which threads into the nanopore (i.e. the Yadaptor end of a RTC polynucleotide).

The inventors have also surprisingly shown that the bias towards doubleY polynucleotides can be overcome by using a Y adaptor and a hairpinloop adaptor which have different strength of coupling to the membrane.If the hairpin loop adaptor couples with more strength than the Yadaptor, a bias towards the RTC polynucleotides is achieved. The“strong” hairpin loop adaptor effectively couples the RTCpolynucleotides to the membrane and this allows them to out compete thedouble Y adaptors which contain two “weaker” tethers. High throughput ismaintained for the RTC polynucleotides because of the “weaker” Yadaptor.

The invention concerns characterising a target double strandedpolynucleotide using a transmembrane pore in a membrane. The targetdouble stranded polynucleotide is provided with a Y adaptor at one endand a hairpin loop adaptor at the other end. Methods of doing this arediscussed in detail below. The Y adaptor comprises one or more firstanchors for coupling the polynucleotide to the membrane and the hairpinloop adaptor comprises one or more second anchors for coupling thepolynucleotide to the membrane. The strength of coupling of the hairpinloop adaptor to the membrane is greater than the strength of coupling ofthe Y adaptor to the membrane.

The polynucleotide provided with the two adaptors is contacted with thetransmembrane pore such that at least one strand of the polynucleotidemoves through the pore. One or more measurements are taken as the atleast one strand of the polynucleotide moves with respect to the pore.The measurements are indicative of one or more characteristics of the atleast one strand of the polynucleotide.

The invention preferably concerns sequencing or estimating the sequenceof a target double stranded polynucleotide. Only part of the targetdouble stranded polynucleotide may be characterised or sequenced asdiscussed in more detail below.

Target Double Stranded Polynucleotide

The method of the invention characterises a target double strandedpolynucleotide. The target double stranded polynucleotide may also becalled the template double stranded polynucleotide or the doublestranded polynucleotide of interest.

A polynucleotide, such as a nucleic acid, is a macromolecule comprisingtwo or more nucleotides. The polynucleotide or nucleic acid may compriseany combination of any nucleotides. The nucleotides can be naturallyoccurring or artificial. One or more nucleotides in the targetpolynucleotide can be oxidized or methylated. One or more nucleotides inthe target polynucleotide may be damaged. For instance, thepolynucleotide may comprise a pyrimidine dimer. Such dimers aretypically associated with damage by ultraviolet light and are theprimary cause of skin melanomas. One or more nucleotides in the targetpolynucleotide may be modified, for instance with a label or a tag.Suitable labels are described below. The target polynucleotide maycomprise one or more spacers.

A nucleotide typically contains a nucleobase, a sugar and at least onephosphate group. The nucleobase and sugar form a nucleoside. Thenucleobase is typically heterocyclic. Nucleobases include, but are notlimited to, purines and pyrimidines and more specifically adenine (A),guanine (G), thymine (T), uracil (U) and cytosine (C).

The sugar is typically a pentose sugar. Nucleotide sugars include, butare not limited to, ribose and deoxyribose. The sugar is preferably adeoxyribose.

The target double stranded polynucleotide preferably comprises thefollowing nucleosides: deoxyadenosine (dA), deoxyuridine (dU) and/orthymidine (dT), deoxyguanosine (dG) and deoxycytidine (dC).

The nucleotide is typically a ribonucleotide or deoxyribonucleotide. Thenucleotide is preferably a deoxyribonucleotide. The nucleotide typicallycontains a monophosphate, diphosphate or triphosphate. Phosphates may beattached on the 5′ or 3′ side of a nucleotide.

Nucleotides include, but are not limited to, adenosine monophosphate(AMP), guanosine monophosphate (GMP), thymidine monophosphate (TMP),uridine monophosphate (UMP), 5-methylcytidine monophosphate,5-hydroxymethylcytidine monophosphate, cytidine monophosphate (CMP),cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate(cGMP), deoxyadenosine monophosphate (dAMP), deoxyguanosinemonophosphate (dGMP), deoxythymidine monophosphate (dTMP), deoxyuridinemonophosphate (dUMP) and deoxycytidine monophosphate (dCMP). Thenucleotides are preferably selected from AMP, TMP, GMP, CMP, UMP, dAMP,dTMP, dGMP, dCMP and dUMP. The nucleotides are most preferably selectedfrom dAMP, dTMP, dGMP, dCMP and dUMP.

The target double stranded polynucleotide preferably comprises thefollowing nucleotides: dAMP, dUMP and/or dTMP, dGMP and dCMP.

A nucleotide may be abasic (i.e. lack a nucleobase). A nucleotide mayalso lack a nucleobase and a sugar (i.e. is a C3 spacer).

The nucleotides in the target polynucleotide may be attached to eachother in any manner. The nucleotides are typically attached by theirsugar and phosphate groups as in nucleic acids. The nucleotides may beconnected via their nucleobases as in pyrimidine dimers.

The target polynucleotide is double stranded. The target polynucleotidemay contain some single stranded regions, but at least a portion of thetarget polynucleotide is double stranded.

The target polynucleotide can be a nucleic acid, such asdeoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The targetpolynucleotide can comprise one strand of RNA hybridised to one strandof DNA. The polynucleotide may be any synthetic nucleic acid known inthe art, such as peptide nucleic acid (PNA), glycerol nucleic acid(GNA), threose nucleic acid (TNA), locked nucleic acid (LNA), bridgednucleic acid (BNA) or other synthetic polymers with nucleotide sidechains. The PNA backbone is composed of repeatingN-(2-aminoethyl)-glycine units linked by peptide bonds. The GNA backboneis composed of repeating glycol units linked by phosphodiester bonds.The TNA backbone is composed of repeating threose sugars linked togetherby phosphodiester bonds. LNA is formed from ribonucleotides as discussedabove having an extra bridge connecting the 2′ oxygen and 4′ carbon inthe ribose moiety. Bridged nucleic acids (BNAs) are modified RNAnucleotides. They may also be called constrained or inaccessible RNA.BNA monomers can contain a five-membered, six-membered or even aseven-membered bridged structure with a “fixed” C3′-endo sugarpuckering. The bridge is synthetically incorporated at the 2′,4′-position of the ribose to produce a 2′, 4′-BNA monomer.

The polynucleotide is most preferably ribonucleic nucleic acid (RNA) ordeoxyribonucleic acid (DNA).

The target polynucleotide can be any length. For example, thepolynucleotide can be at least 10, at least 50, at least 100, at least150, at least 200, at least 250, at least 300, at least 400 or at least500 nucleotide pairs in length. The polynucleotide can be 1000 or morenucleotide pairs, 5000 or more nucleotide pairs in length or 100000 ormore nucleotide pairs in length.

The target polynucleotide is typically present in any suitable sample.The invention is typically carried out on a sample that is known tocontain or suspected to contain the target polynucleotide.Alternatively, the invention may be carried out on a sample to confirmthe identity of one or more target polynucleotides whose presence in thesample is known or expected.

The sample may be a biological sample. The invention may be carried outin vitro on a sample obtained from or extracted from any organism ormicroorganism. The organism or microorganism is typically archaeal,prokaryotic or eukaryotic and typically belongs to one of the fivekingdoms: plantae, animalia, fungi, monera and protista. The inventionmay be carried out in vitro on a sample obtained from or extracted fromany virus. The sample is preferably a fluid sample. The sample typicallycomprises a body fluid of the patient. The sample may be urine, lymph,saliva, mucus or amniotic fluid but is preferably blood, plasma orserum. Typically, the sample is human in origin, but alternatively itmay be from another mammal animal such as from commercially farmedanimals such as horses, cattle, sheep, fish, chickens or pigs or mayalternatively be pets such as cats or dogs. Alternatively, the samplemay be of plant origin, such as a sample obtained from a commercialcrop, such as a cereal, legume, fruit or vegetable, for example wheat,barley, oats, canola, maize, soya, rice, rhubarb, bananas, apples,tomatoes, potatoes, grapes, tobacco, beans, lentils, sugar cane, cocoa,cotton.

The sample may be a non-biological sample. The non-biological sample ispreferably a fluid sample. Examples of a non-biological sample includesurgical fluids, water such as drinking water, sea water or river water,and reagents for laboratory tests.

The sample is typically processed prior to being used in the invention,for example by centrifugation or by passage through a membrane thatfilters out unwanted molecules or cells, such as red blood cells. Thesample may be measured immediately upon being taken. The sample may alsobe typically stored prior to assay, preferably below −70° C.

Providing the Polynucleotide with Two Adaptors

The target double stranded polynucleotide is provided with a Y adaptorat one end and a hairpin loop adaptor at the other end. The Y adaptorand/or the hairpin loop adaptor are typically polynucleotide adaptors.They may be formed from any of the polynucleotides discussed above.

The Y adaptor typically comprises a double stranded region and a singlestranded region or a region that is not complementary at the other end.The Y adaptor may be described as having an overhang if it comprises asingle stranded region. The presence of a non-complementary region inthe Y adaptor gives the adaptor its Y shape since the two strandstypically do not hybridise to each other unlike the double strandedportion. The Y adaptor comprises the one or more first anchors. Anchorsare discussed in more detail below.

The Y adaptor preferably comprises a leader sequence whichpreferentially threads into the pore. The leader sequence facilitatesthe method of the invention. The leader sequence is designed topreferentially thread into the transmembrane pore and thereby facilitatethe movement of polynucleotide through the pore. The leader sequence canalso be used to link the polynucleotide to the anchor(s) as discussedbelow.

The leader sequence typically comprises a polymer. The polymer ispreferably negatively charged. The polymer is preferably apolynucleotide, such as DNA or RNA, a modified polynucleotide (such asabasic DNA), PNA, LNA, polyethylene glycol (PEG) or a polypeptide. Theleader preferably comprises a polynucleotide and more preferablycomprises a single stranded polynucleotide. The leader sequence cancomprise any of the polynucleotides discussed above. The single strandedleader sequence most preferably comprises a single strand of DNA, suchas a poly dT section. The leader sequence preferably comprises the oneor more spacers.

The leader sequence can be any length, but is typically 10 to 150nucleotides in length, such as from 20 to 150 nucleotides in length. Thelength of the leader typically depends on the transmembrane pore used inthe method.

Suitable hairpin loop adaptors can be designed using methods known inthe art. The hairpin loop may be any length. The hairpin loop istypically 110 or fewer nucleotides, such as 100 or fewer nucleotides, 90or fewer nucleotides, 80 or fewer nucleotides, 70 or fewer nucleotides,60 or fewer nucleotides, 50 or fewer nucleotides, 40 or fewernucleotides, 30 or fewer nucleotides, 20 or fewer nucleotides or 10 orfewer nucleotides, in length. The hairpin loop is preferably from about1 to 110, from 2 to 100, from 5 to 80 or from 6 to 50 nucleotides inlength. Longer lengths of the hairpin loop, such as from 50 to 110nucleotides, are preferred if the loop is involved in the differentialselectability of the adaptor. Similarly, shorter lengths of the hairpinloop, such as from 1 to 5 nucleotides, are preferred if the loop is notinvolved in the selectable binding as discussed below.

The hairpin loop adaptor preferably comprises a selectable bindingmoiety. This allows the first and/or second polynucleotide to bepurified or isolated. A selectable binding moiety is a moiety that canbe selected on the basis of its binding properties. Hence, a selectablebinding moiety is preferably a moiety that specifically binds to asurface. A selectable binding moiety specifically binds to a surface ifit binds to the surface to a much greater degree than any other moietyused in the invention. In preferred embodiments, the moiety binds to asurface to which no other moiety used in the invention binds.

Suitable selective binding moieties are known in the art. Preferredselective binding moieties include, but are not limited to, biotin, apolynucleotide sequence, antibodies, antibody fragments, such as Fab andScSv, antigens, polynucleotide binding proteins, poly histidine tailsand GST tags. The most preferred selective binding moieties are biotinand a selectable polynucleotide sequence. Biotin specifically binds to asurface coated with avidins. Selectable polynucleotide sequencesspecifically bind (i.e. hybridise) to a surface coated with homologussequences. Alternatively, selectable polynucleotide sequencesspecifically bind to a surface coated with polynucleotide bindingproteins.

The hairpin loop adaptor and/or the selectable binding moiety maycomprise a region that can be cut, nicked, cleaved or hydrolysed. Such aregion can be designed to allow the first and/or second polynucleotideto be removed from the surface to which it is bound followingpurification or isolation. Suitable regions are known in the art.Suitable regions include, but are not limited to, an RNA region, aregion comprising desthiobiotin and streptavidin, a disulphide bond anda photocleavable region.

The Y adaptor and/or the hairpin loop adaptor may be ligated to thepolynucleotide using any method known in the art. One or both of theadaptors may be ligated using a ligase, such as T4 DNA ligase, E. coliDNA ligase, Taq DNA ligase, Tma DNA ligase and 9° N DNA ligase.Alternatively, the adaptors may be added to the polynucleotide using themethods of the invention discussed below.

In a preferred embodiment, step a) of the method comprises modifying thetarget double stranded polynucleotide so that it comprises the Y adaptorat one end and the hairpin loop adaptor at the other end. Any manner ofmodification can be used. The method preferably comprises modifying thetarget double stranded polynucleotide in accordance with the invention.This is discussed in more detail below. The methods of modification andcharacterisation may be combined in any way.

Membrane

Any membrane may be used in accordance with the invention. Suitablemembranes are well-known in the art. The membrane is preferably anamphiphilic layer. An amphiphilic layer is a layer formed fromamphiphilic molecules, such as phospholipids, which have bothhydrophilic and lipophilic properties. The amphiphilic molecules may besynthetic or naturally occurring. Non-naturally occurring amphiphilesand amphiphiles which form a monolayer are known in the art and include,for example, block copolymers (Gonzalez-Perez et al., Langmuir, 2009,25, 10447-10450). Block copolymers are polymeric materials in which twoor more monomer sub-units that are polymerized together to create asingle polymer chain. Block copolymers typically have properties thatare contributed by each monomer sub-unit. However, a block copolymer mayhave unique properties that polymers formed from the individualsub-units do not possess. Block copolymers can be engineered such thatone of the monomer sub-units is hydrophobic (i.e. lipophilic), whilstthe other sub-unit(s) are hydrophilic whilst in aqueous media. In thiscase, the block copolymer may possess amphiphilic properties and mayform a structure that mimics a biological membrane. The block copolymermay be a diblock (consisting of two monomer sub-units), but may also beconstructed from more than two monomer sub-units to form more complexarrangements that behave as amphipiles. The copolymer may be a triblock,tetrablock or pentablock copolymer. The membrane is preferably atriblock copolymer membrane.

Archaebacterial bipolar tetraether lipids are naturally occurring lipidsthat are constructed such that the lipid forms a monolayer membrane.These lipids are generally found in extremophiles that survive in harshbiological environments, thermophiles, halophiles and acidophiles. Theirstability is believed to derive from the fused nature of the finalbilayer. It is straightforward to construct block copolymer materialsthat mimic these biological entities by creating a triblock polymer thathas the general motif hydrophilic-hydrophobic-hydrophilic. This materialmay form monomeric membranes that behave similarly to lipid bilayers andencompass a range of phase behaviours from vesicles through to laminarmembranes. Membranes formed from these triblock copolymers hold severaladvantages over biological lipid membranes. Because the triblockcopolymer is synthesized, the exact construction can be carefullycontrolled to provide the correct chain lengths and properties requiredto form membranes and to interact with pores and other proteins.

Block copolymers may also be constructed from sub-units that are notclassed as lipid sub-materials; for example a hydrophobic polymer may bemade from siloxane or other non-hydrocarbon based monomers. Thehydrophilic sub-section of block copolymer can also possess low proteinbinding properties, which allows the creation of a membrane that ishighly resistant when exposed to raw biological samples. This head groupunit may also be derived from non-classical lipid head-groups.

Triblock copolymer membranes also have increased mechanical andenvironmental stability compared with biological lipid membranes, forexample a much higher operational temperature or pH range. The syntheticnature of the block copolymers provides a platform to customize polymerbased membranes for a wide range of applications.

In a preferred embodiment, the invention provides a method ofcharacterising a target double stranded polynucleotide using atransmembrane pore in a membrane comprising a triblock copolymer,optionally wherein the membrane is modified to facilitate the coupling.

The membrane is most preferably one of the membranes disclosed inInternational Application No. PCT/GB2013/052766 or PCT/GB2013/052767.

The amphiphilic molecules may be chemically-modified or functionalisedto facilitate coupling of the polynucleotide.

The amphiphilic layer may be a monolayer or a bilayer. The amphiphiliclayer is typically planar. The amphiphilic layer may be curved. Theamphiphilic layer may be supported.

Amphiphilic membranes are typically naturally mobile, essentially actingas two dimensional fluids with lipid diffusion rates of approximately10⁻⁸ cm s⁻¹. This means that the pore and coupled polynucleotide cantypically move within an amphiphilic membrane.

The membrane may be a lipid bilayer. Lipid bilayers are models of cellmembranes and serve as excellent platforms for a range of experimentalstudies. For example, lipid bilayers can be used for in vitroinvestigation of membrane proteins by single-channel recording.Alternatively, lipid bilayers can be used as biosensors to detect thepresence of a range of substances. The lipid bilayer may be any lipidbilayer. Suitable lipid bilayers include, but are not limited to, aplanar lipid bilayer, a supported bilayer or a liposome. The lipidbilayer is preferably a planar lipid bilayer. Suitable lipid bilayersare disclosed in International Application No. PCT/GB08/000563(published as WO 2008/102121), International Application No.PCT/GB08/004127 (published as WO 2009/077734) and InternationalApplication No. PCT/GB2006/001057 (published as WO 2006/100484).

Methods for forming lipid bilayers are known in the art. Suitablemethods are disclosed in the Example. Lipid bilayers are commonly formedby the method of Montal and Mueller (Proc. Natl. Acad. Sci. USA., 1972;69: 3561-3566), in which a lipid monolayer is carried on aqueoussolution/air interface past either side of an aperture which isperpendicular to that interface. The lipid is normally added to thesurface of an aqueous electrolyte solution by first dissolving it in anorganic solvent and then allowing a drop of the solvent to evaporate onthe surface of the aqueous solution on either side of the aperture. Oncethe organic solvent has evaporated, the solution/air interfaces oneither side of the aperture are physically moved up and down past theaperture until a bilayer is formed. Planar lipid bilayers may be formedacross an aperture in a membrane or across an opening into a recess.

The method of Montal & Mueller is popular because it is a cost-effectiveand relatively straightforward method of forming good quality lipidbilayers that are suitable for protein pore insertion. Other commonmethods of bilayer formation include tip-dipping, painting bilayers andpatch-clamping of liposome bilayers.

Tip-dipping bilayer formation entails touching the aperture surface (forexample, a pipette tip) onto the surface of a test solution that iscarrying a monolayer of lipid. Again, the lipid monolayer is firstgenerated at the solution/air interface by allowing a drop of lipiddissolved in organic solvent to evaporate at the solution surface. Thebilayer is then formed by the Langmuir-Schaefer process and requiresmechanical automation to move the aperture relative to the solutionsurface.

For painted bilayers, a drop of lipid dissolved in organic solvent isapplied directly to the aperture, which is submerged in an aqueous testsolution. The lipid solution is spread thinly over the aperture using apaintbrush or an equivalent. Thinning of the solvent results information of a lipid bilayer. However, complete removal of the solventfrom the bilayer is difficult and consequently the bilayer formed bythis method is less stable and more prone to noise duringelectrochemical measurement.

Patch-clamping is commonly used in the study of biological cellmembranes. The cell membrane is clamped to the end of a pipette bysuction and a patch of the membrane becomes attached over the aperture.The method has been adapted for producing lipid bilayers by clampingliposomes which then burst to leave a lipid bilayer sealing over theaperture of the pipette. The method requires stable, giant andunilamellar liposomes and the fabrication of small apertures inmaterials having a glass surface.

Liposomes can be formed by sonication, extrusion or the Mozafari method(Colas et al. (2007) Micron 38: 841-847).

In a preferred embodiment, the lipid bilayer is formed as described inInternational Application No. PCT/GB08/004127 (published as WO2009/077734). Advantageously in this method, the lipid bilayer is formedfrom dried lipids. In a most preferred embodiment, the lipid bilayer isformed across an opening as described in W02009/077734(PCT/GB08/004127).

A lipid bilayer is formed from two opposing layers of lipids. The twolayers of lipids are arranged such that their hydrophobic tail groupsface towards each other to form a hydrophobic interior. The hydrophilichead groups of the lipids face outwards towards the aqueous environmenton each side of the bilayer. The bilayer may be present in a number oflipid phases including, but not limited to, the liquid disordered phase(fluid lamellar), liquid ordered phase, solid ordered phase (lamellargel phase, interdigitated gel phase) and planar bilayer crystals(lamellar sub-gel phase, lamellar crystalline phase).

Any lipid composition that forms a lipid bilayer may be used. The lipidcomposition is chosen such that a lipid bilayer having the requiredproperties, such surface charge, ability to support membrane proteins,packing density or mechanical properties, is formed. The lipidcomposition can comprise one or more different lipids. For instance, thelipid composition can contain up to 100 lipids. The lipid compositionpreferably contains 1 to 10 lipids. The lipid composition may comprisenaturally-occurring lipids and/or artificial lipids. The lipidstypically comprise a head group, an interfacial moiety and twohydrophobic tail groups which may be the same or different. Suitablehead groups include, but are not limited to, neutral head groups, suchas diacylglycerides (DG) and ceramides (CM); zwitterionic head groups,such as phosphatidylcholine (PC), phosphatidylethanolamine (PE) andsphingomyelin (SM); negatively charged head groups, such asphosphatidylglycerol (PG); phosphatidylserine (PS), phosphatidylinositol(PI), phosphatic acid (PA) and cardiolipin (CA); and positively chargedheadgroups, such as trimethylammonium-Propane (TAP). Suitableinterfacial moieties include, but are not limited to,naturally-occurring interfacial moieties, such as glycerol-based orceramide-based moieties. Suitable hydrophobic tail groups include, butare not limited to, saturated hydrocarbon chains, such as lauric acid(n-Dodecanolic acid), myristic acid (n-Tetradecononic acid), palmiticacid (n-Hexadecanoic acid), stearic acid (n-Octadecanoic) and arachidic(n-Eicosanoic); unsaturated hydrocarbon chains, such as oleic acid(cis-9-Octadecanoic); and branched hydrocarbon chains, such asphytanoyl. The length of the chain and the position and number of thedouble bonds in the unsaturated hydrocarbon chains can vary. The lengthof the chains and the position and number of the branches, such asmethyl groups, in the branched hydrocarbon chains can vary. Thehydrophobic tail groups can be linked to the interfacial moiety as anether or an ester. The lipids may be mycolic acid.

The lipids can also be chemically-modified. The head group or the tailgroup of the lipids may be chemically-modified. Suitable lipids whosehead groups have been chemically-modified include, but are not limitedto, PEG-modified lipids, such as1,2-Diacyl-sn-Glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethyleneglycol)-2000]; functionalised PEG Lipids, such as1,2-Distearoyl-sn-Glycero-3 Phosphoethanolamine-N-[Biotinyl(PolyethyleneGlycol)2000]; and lipids modified for conjugation, such as1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine-N-(succinyl) and1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine-N-(Biotinyl). Suitablelipids whose tail groups have been chemically-modified include, but arenot limited to, polymerisable lipids, such as1,2-bis(10,12-tricosadiynoyl)-sn-Glycero-3-Phosphocholine; fluorinatedlipids, such as1-Palmitoyl-2-(16-Fluoropalmitoyl)-sn-Glycero-3-Phosphocholine;deuterated lipids, such as1,2-Dipalmitoyl-D62-sn-Glycero-3-Phosphocholine; and ether linkedlipids, such as 1,2-Di-O-phytanyl-sn-Glycero-3-Phosphocholine. Thelipids may be chemically-modified or functionalised to facilitatecoupling of the polynucleotide.

The amphiphilic layer, for example the lipid composition, typicallycomprises one or more additives that will affect the properties of thelayer. Suitable additives include, but are not limited to, fatty acids,such as palmitic acid, myristic acid and oleic acid; fatty alcohols,such as palmitic alcohol, myristic alcohol and oleic alcohol; sterols,such as cholesterol, ergosterol, lanosterol, sitosterol andstigmasterol; lysophospholipids, such as1-Acyl-2-Hydroxy-sn-Glycero-3-Phosphocholine; and ceramides.

In another preferred embodiment, the membrane is a solid state layer.Solid state layers can be formed from both organic and inorganicmaterials including, but not limited to, microelectronic materials,insulating materials such as Si₃N_(4,) Al₂O_(3,) and SiO, organic andinorganic polymers such as polyamide, plastics such as Teflon® orelastomers such as two-component addition-cure silicone rubber, andglasses. The solid state layer may be formed from graphene. Suitablegraphene layers are disclosed in International Application No.PCT/US2008/010637 (published as WO 2009/035647).

The method is typically carried out using (i) an artificial amphiphiliclayer comprising a pore, (ii) an isolated, naturally-occurring lipidbilayer comprising a pore, or (iii) a cell having a pore insertedtherein. The method is typically carried out using an artificialamphiphilic layer, such as an artificial triblock copolymer layer. Thelayer may comprise other transmembrane and/or intramembrane proteins aswell as other molecules in addition to the pore. Suitable apparatus andconditions are discussed below. The method of the invention is typicallycarried out in vitro.

Coupling

The polynucleotide may be coupled to the membrane using any knownmethod. The polynucleotide is coupled to the membrane using at least twoanchors.

The Y adaptor comprises one or more first anchors. The hairpin loopadaptor comprises one or more second adaptors. Each anchor comprises agroup which couples (or binds) to the adaptor and a group which couples(or binds) to the membrane. Each anchor may covalently couple (or bind)to the adaptor and/or the membrane.

The Y adaptor may contain any number of first anchors, such as 2, 3, 4or more anchors. The hairpin loop adaptor may contain any number ofsecond anchors, such as 2, 3, 4 or more anchors. For instance, one orboth adaptors may comprise two anchors each of which separately couples(or binds) to both the adaptor(s) and membrane.

The one or more first anchors and/or the one or more second anchors maycomprise one or more polynucleotide binding proteins. Each anchor maycomprise one or more polynucleotide binding proteins. The polynucleotidebinding protein(s) may be any of those discussed above.

If the membrane is an amphiphilic layer, such as a copolymer membrane ora lipid bilayer, the one or more anchors preferably comprise apolypeptide anchor present in the membrane and/or a hydrophobic anchorpresent in the membrane. The hydrophobic anchor is preferably a lipid,fatty acid, sterol, carbon nanotube, polypeptide, protein or amino acid,for example cholesterol, palmitate or tocopherol. In preferredembodiments, the one or more anchors are not the detector.

The components of the membrane, such as the amphiphilic molecules,copolymer or lipids, may be chemically-modified or functionalised toform the one or more anchors. Examples of suitable chemicalmodifications and suitable ways of functionalising the components of themembrane are discussed in more detail below. Any proportion of themembrane components may be functionalized, for example at least 0.01%,at least 0.1%, at least 1%, at least 10%, at least 25%, at least 50% or100%.

The Y adaptor and/or the hairpin loop adaptor may be coupled directly tothe membrane. The one or more first anchors and/or the one or moresecond anchors preferably comprise a linker. The one or more firstanchors and/or the one or more second anchors may comprise one or more,such as 2, 3, 4 or more, linkers. One linker may be used to couple morethan one, such as 2, 3, 4 or more, polynucleotides or adaptors to themembrane.

Preferred linkers include, but are not limited to, polymers, such aspolynucleotides, polyethylene glycols (PEGs), polysaccharides andpolypeptides. These linkers may be linear, branched or circular. Forinstance, the linker may be a circular polynucleotide. Thepolynucleotide to be characterised may hybridise to a complementarysequence on the circular polynucleotide linker.

The one or more anchors or one or more linkers may comprise a componentthat can be cut or broken down, such as a restriction site or aphotolabile group.

Functionalised linkers and the ways in which they can couple moleculesare known in the art. For instance, linkers functionalised withmaleimide groups will react with and attach to cysteine residues inproteins. In the context of this invention, the protein may be presentin the membrane or may be used to couple (or bind) to thepolynucleotide. This is discussed in more detail below. Crosslinkage ofpolynucleotides can be avoided using a “lock and key” arrangement.

Only one end of each linker may react together to form a longer linkerand the other ends of the linker each react with the polynucleotide ormembrane respectively. Such linkers are described in InternationalApplication No. PCT/GB10/000132 (published as WO 2010/086602).

The use of a linker is preferred in the sequencing embodiments discussedbelow. If a polynucleotide is permanently coupled directly to themembrane in the sense that it does not uncouple when interacting withthe pore, then some sequence data will be lost as the sequencing runcannot continue to the end of the polynucleotide due to the distancebetween the membrane and the detector. If a linker is used, then thepolynucleotide can be processed to completion.

The coupling may be permanent or stable. In other words, the couplingmay be such that the polynucleotide remains coupled to the membrane wheninteracting with the pore.

The coupling may be transient. In other words, the coupling may be suchthat the polynucleotide may decouple from the membrane when interactingwith the pore.

Chemical groups that form permanent/stable or transient links arediscussed in more detail below. The polynucleotide may be transientlycoupled to an amphiphilic layer or triblock copolymer membrane usingcholesterol or a fatty acyl chain. Any fatty acyl chain having a lengthof from 6 to 30 carbon atom, such as hexadecanoic acid, may be used.

In preferred embodiments, a polynucleotide, such as a nucleic acid, iscoupled to an amphiphilic layer such as a triblock copolymer membrane orlipid bilayer. Coupling of nucleic acids to synthetic lipid bilayers hasbeen carried out previously with various different tethering strategies.These are summarised in Table 1 below.

TABLE 1 Anchor Type of comprising coupling Reference Thiol StableYoshina-Ishii, C. and S. G. Boxer (2003). “Arrays of mobile tetheredvesicles on supported lipid bilayers.” J Am Chem Soc 125(13): 3696-7.Biotin Stable Nikolov, V., R. Lipowsky, et al. (2007). “Behavior ofgiant vesicles with anchored DNA molecules.” Biophys J 92(12): 4356-68Cholesterol Transient Pfeiffer, I. and F. Hook (2004). “Bivalentcholesterol-based coupling of oligonucletides to lipid membraneassemblies.” J Am Chem Soc 126(33): 10224-5 Surfactant Stable vanLengerich, B., R. J. Rawle, et al. (e.g. Lipid, “Covalent attachment oflipid vesicles to a Palmitate, fluid-supported bilayer allowsobservation of etc) DNA-mediated vesicle interactions.” Langmuir 26(11):8666-72

Synthetic polynucleotides and/or linkers may be functionalised using amodified phosphoramidite in the synthesis reaction, which is easilycompatible for the direct addition of suitable anchoring groups, such ascholesterol, tocopherol, palmitate, thiol, lipid and biotin groups.These different attachment chemistries give a suite of options forattachment to polynucleotides. Each different modification group couplesthe polynucleotide in a slightly different way and coupling is notalways permanent so giving different dwell times for the polynucleotideto the membrane. The advantages of transient coupling are discussedabove.

Coupling of polynucleotides to a linker or to a functionalised membranecan also be achieved by a number of other means provided that acomplementary reactive group or an anchoring group can be added to thepolynucleotide. The addition of reactive groups to either end of apolynucleotide has been reported previously. A thiol group can be addedto the 5′ of ssDNA or dsDNA using T4 polynucleotide kinase and ATPγS(Grant, G. P. and P. Z. Qin (2007). “A facile method for attachingnitroxide spin labels at the 5′ terminus of nucleic acids.” NucleicAcids Res 35(10): e77). An azide group can be added to the 5′-phosphateof ssDNA or dsDNA using T4 polynucleotide kinase andγ-[2-Azidoethyl]-ATP or γ-[6-Azidohexyl]-ATP. Using thiol or Clickchemistry a tether, containing either a thiol, iodoacetamide OPSS ormaleimide group (reactive to thiols) or a DIBO (dibenzocyclooxtyne) oralkyne group (reactive to azides), can be covalently attached to thepolynucleotide. A more diverse selection of chemical groups, such asbiotin, thiols and fluorophores, can be added using terminal transferaseto incorporate modified oligonucleotides to the 3′ of ssDNA (Kumar, A.,P. Tchen, et al. (1988). “Nonradioactive labeling of syntheticoligonucleotide probes with terminal deoxynucleotidyl transferase.”AnalBiochem 169(2): 376-82). Streptavidin/biotin and/orstreptavidin/desthiobiotin coupling may be used for polynucleotide. Itmay also be possible that anchors may be directly added topolynucleotides using terminal transferase with suitably modifiednucleotides (e.g. cholesterol or palmitate).

The one or more first anchors and/or the one or more second anchorspreferably couple the target double stranded polynucleotide to themembrane via hybridisation. The hybridisation may be present in any partof the one or more anchors, such as between the one or more anchors andthe polynucleotide, within the one or more anchors or between the one ormore anchors and the membrane. Hybridisation in the one or more anchorsallows coupling in a transient manner as discussed above. For instance,a linker may comprise two or more polynucleotides, such as 3, 4 or 5polynucleotides, hybridised together. The one or more first anchorsand/or the one or more second anchors may hybridise to the target doublestranded polynucleotide. The one or more first anchors may hybridisedirectly to the target double stranded polynucleotide or directly to theY adaptor and/or leader sequence. The one or more second anchors mayhybridise directly to the target double stranded polynucleotide ordirectly to the hairpin adaptor. Alternatively, the one or more firstanchors may be hybridised to one or more, such as 2 or 3, intermediatepolynucleotides (or “splints”) which are hybridised to the target doublestranded polynucleotide or to the Y adaptor and/or leader sequence.Alternatively, the one or more second anchors may be hybridised to oneor more, such as 2 or 3, intermediate polynucleotides (or “splints”)which are hybridised to the target double stranded polynucleotide or tothe hairpin loop adaptor. The hybridisation of the one or more anchorsto one or more splints may form one or more rigid double strandedpolynucleotide linkers as discussed in more detail below.

The one or more first anchors and/or the one or more second anchors maycomprise a single stranded or double stranded polynucleotide. One partof the anchor may be ligated to the double stranded polynucleotide.Ligation of short pieces of ssDNA have been reported using T4 RNA ligaseI (Troutt, A. B., M. G. McHeyzer-Williams, et al. (1992).“Ligation-anchored PCR: a simple amplification technique withsingle-sided specificity.” Proc Natl Acad Sci U S A 89(20): 9823-5).Alternatively, either a single stranded or double strandedpolynucleotide can be ligated to a double stranded polynucleotide andthen the two strands separated by thermal or chemical denaturation. To adouble stranded polynucleotide, it is possible to add either a piece ofsingle stranded polynucleotide to one or both of the ends of the duplex,or a double stranded polynucleotide to one or both ends. For addition ofsingle stranded polynucleotides to the a double stranded polynucleotide,this can be achieved using T4 RNA ligase I as for ligation to otherregions of single stranded polynucleotides. For addition of doublestranded polynucleotides to a double stranded polynucleotide thenligation can be “blunt-ended”, with complementary 3′ dA/dT tails on thetarget and added polynucleotides respectively (as is routinely done formany sample prep applications to prevent concatemer or dimer formation)or using “sticky-ends” generated by restriction digestion of thepolynucleotide and ligation of compatible adapters. Then, when theduplex is melted, each single strand will have either a 5′ or 3′modification if a single stranded polynucleotide was used for ligationor a modification at the 5′ end, the 3′ end or both if a double strandedpolynucleotide was used for ligation.

If the polynucleotide is a synthetic strand, the adaptors and anchorscan be incorporated during the chemical synthesis of the polynucleotide.For instance, the polynucleotide can be synthesised using a primerhaving a reactive group attached to it.

Adenylated polynucleotides are intermediates in ligation reactions,where an adenosine-monophosphate is attached to the 5′-phosphate of thepolynucleotide. Various kits are available for generation of thisintermediate, such as the 5′ DNA Adenylation Kit from NEB. Bysubstituting ATP in the reaction for a modified nucleotide triphosphate,then addition of reactive groups (such as thiols, amines, biotin,azides, etc) to the 5′ of a polynucleotide can be possible.

It may also be possible that anchors could be directly added topolynucleotides using a 5′ DNA adenylation kit with suitably modifiednucleotides (e.g. cholesterol or palmitate).

A common technique for the amplification of sections of genomic DNA isusing polymerase chain reaction (PCR). Here, using two syntheticoligonucleotide primers, a number of copies of the same section of DNAcan be generated, where for each copy the 5′ of each strand in theduplex will be a synthetic polynucleotide. Single or multiplenucleotides can be added to 3′ end of single or double stranded DNA byemploying a polymerase. Examples of polymerases which could be usedinclude, but are not limited to, Terminal Transferase, Klenow and E.coli Poly(A) polymerase). By substituting dATP in the reaction for amodified nucleotide triphosphate then anchors, such as cholesterol,thiol, amine, azide, biotin or lipid, can be incorporated into doublestranded polynucleotides. Therefore, each copy of the amplifiedpolynucleotide will contain an anchor.

The anchor can comprise any group that couples to, binds to or interactswith single or double stranded polynucleotides, specific nucleotidesequences within the polynucleotide or patterns of modified nucleotideswithin the polynucleotide, or any other ligand that is present on thepolynucleotide.

Suitable polynucleotide binding proteins for use in anchors include, butare not limited to, E. coli single stranded binding protein, P5 singlestranded binding protein, T4 gp32 single stranded binding protein, theTOPO V dsDNA binding region, human histone proteins, E. coli HU DNAbinding protein and other archaeal, prokaryotic or eukaryotic singlestranded or double stranded polynucleotide (or nucleic acid) bindingproteins, including those listed below.

The specific nucleotide sequences could be sequences recognised bytranscription factors, ribosomes, endonucleases, topoisomerases orreplication initiation factors. The patterns of modified nucleotidescould be patterns of methylation or damage.

The one or more first anchors and/or the one or more second anchors cancomprise any group which binds to, intercalates with or interacts with apolynucleotide. The group may intercalate or interact with thepolynucleotide via electrostatic, hydrogen bonding or Van der Waalsinteractions. Such groups include a lysine monomer, poly-lysine (whichwill interact with ssDNA or dsDNA), ethidium bromide (which willintercalate with dsDNA), universal bases or universal nucleotides (whichcan hybridise with any polynucleotide) and osmium complexes (which canreact to methylated bases). A polynucleotide may therefore be coupled tothe membrane using one or more universal nucleotides attached to themembrane. Each universal nucleotide may be coupled to the membrane usingone or more linkers. The universal nucleotide preferably comprises oneof the following nucleobases: hypoxanthine, 4-nitroindole,5-nitroindole, 6-nitroindole, formylindole, 3-nitropyrrole,nitroimidazole, 4-nitropyrazole, 4-nitrobenzimidazole, 5-nitroindazole,4-aminobenzimidazole or phenyl (C6-aromatic ring). The universalnucleotide more preferably comprises one of the following nucleosides:2′-deoxyinosine, inosine, 7-deaza-2′-deoxyinosine, 7-deaza-inosine,2-aza-deoxyinosine, 2-aza-inosine, 2-O′-methylinosine, 4-nitroindole2′-deoxyribonucleoside, 4-nitroindole ribonucleoside, 5-nitroindole2′-deoxyribonucleoside, 5-nitroindole ribonucleoside, 6-nitroindole2′-deoxyribonucleoside, 6-nitroindole ribonucleoside, 3-nitropyrrole2′-deoxyribonucleoside, 3-nitropyrrole ribonucleoside, an acyclic sugaranalogue of hypoxanthine, nitroimidazole 2′-deoxyribonucleoside,nitroimidazole ribonucleoside, 4-nitropyrazole 2′-deoxyribonucleoside,4-nitropyrazole ribonucleoside, 4-nitrobenzimidazole2′-deoxyribonucleoside, 4-nitrobenzimidazole ribonucleoside,5-nitroindazole 2′-deoxyribonucleoside, 5-nitroindazole ribonucleoside,4-aminobenzimidazole 2′-deoxyribonucleoside, 4-aminobenzimidazoleribonucleoside, phenyl C-ribonucleoside, phenyl C-2′-deoxyribosylnucleoside, 2′-deoxynebularine, 2′-deoxyisoguanosine, K-2′-deoxyribose,P-2′-deoxyribose and pyrrolidine. The universal nucleotide morepreferably comprises 2′-deoxyinosine. The universal nucleotide is morepreferably IMP or dIMP. The universal nucleotide is most preferably dPMP(2′-Deoxy-P-nucleoside monophosphate) or dKMP (N6-methoxy-2,6-diaminopurine monophosphate).

The one or more anchors may couple to (or bind to) the polynucleotidevia Hoogsteen hydrogen bonds (where two nucleobases are held together byhydrogen bonds) or reversed Hoogsteen hydrogen bonds (where onenucleobase is rotated through 180° with respect to the othernucleobase). For instance, the one or more anchors may comprise one ormore nucleotides, one or more oligonucleotides or one or morepolynucleotides which form Hoogsteen hydrogen bonds or reversedHoogsteen hydrogen bonds with the polynucleotide. These types ofhydrogen bonds allow a third polynucleotide strand to wind around adouble stranded helix and form a triplex. The one or more anchors maycouple to (or bind to) a double stranded polynucleotide by forming atriplex with the double stranded duplex.

In this embodiment at least 1%, at least 10%, at least 25%, at least 50%or 100% of the membrane components may be functionalized.

Where the one or more first anchors and/or the one or more secondanchors comprise a protein, it may be able to anchor directly into themembrane without further functonalisation, for example if it already hasan external hydrophobic region which is compatible with the membrane.Examples of such proteins include, but are not limited to, transmembraneproteins, intramembrane proteins and membrane proteins. Alternativelythe protein may be expressed with a genetically fused hydrophobic regionwhich is compatible with the membrane. Such hydrophobic protein regionsare known in the art.

In another aspect the Y adaptor and/or the hairpin loop adaptor may befunctionalised, using methods described above, so that it/they can berecognised by a specific binding group. Specifically the adaptor(s) maybe functionalised with a ligand such as biotin (for binding tostreptavidin), amylose (for binding to maltose binding protein or afusion protein), Ni-NTA (for binding to poly-histidine or poly-histidinetagged proteins) or peptides (such as an antigen).

An example of a molecule used in chemical attachment is EDC(1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride). Reactivegroups can also be added to the 5′ of polynucleotides using commerciallyavailable kits (Thermo Pierce, Part No. 22980). Suitable methodsinclude, but are not limited to, transient affinity attachment usinghistidine residues and Ni-NTA, as well as more robust covalentattachment by reactive cysteines, lysines or non natural amino acids.

Strength of Coupling

The strength of coupling (or binding) of the hairpin loop adaptor to themembrane is greater than the strength of coupling (or binding) of the Yadaptor to the membrane. This can be measured in any way. A suitablemethod for measuring the strength of coupling (or binding) is disclosedin the Examples.

The strength of coupling (or binding) of the hairpin loop adaptor ispreferably at least 1.5 times the strength of coupling (or binding) ofthe Y-adaptor, such as at least twice, at least three times, at leastfour times, at least five or at least ten times the strength of coupling(or binding) of the Y-adaptor. The affinity constant (Kd) of the hairpinloop adaptor for the membrane is preferably at least 1.5 times theaffinity constant of the Y adaptor, such as at least twice, at leastthree times, at least four times, at least five or at least ten timesthe strength of coupling of the Y adaptor.

There are several ways in which the hairpin loop adaptor couples (orbinds) more strongly to the membrane than the Y adaptor. For instance,the hairpin loop adaptor may comprise more anchors than the Y adaptor.For instance, the hairpin loop adaptor may comprise 2, 3 or more secondanchors whereas the Y adaptor may comprise one first anchor. Thestrength of coupling (or binding) of the one or more second anchors tothe membrane may be greater than the strength of coupling (or binding)of the one or more first anchors to the membrane. The strength ofcoupling (or binding) of the one or more second anchors to the hairpinloop adaptor may be greater than the strength of coupling (or binding)of the one or more first anchors to the Y adaptor. The one or more firstanchors and the one or more second anchors may be attached to theirrespective adaptors via hybridisation and the strength of hybridisationis greater in the one or more second anchors than in the one or morefirst anchors. Any combination of these embodiments may also be used inthe invention. Strength of coupling (or binding) may be measure usingknown techniques in the art.

The one or more second anchors preferably comprise one or more groupswhich couples(s) (or bind(s)) to the membrane with a greater strengththan the one or more groups in the one or more first anchors whichcouple(s) (or bind(s)) to the membrane. In preferred embodiments, thehairpin loop adaptor/one or more second anchors couple (or bind) to themembrane using cholesterol and the Y adaptor/one or more first anchorscouple (or bind) to the membrane using palmitate. Cholesterol binds totriblock copolymer membranes and lipid membranes more strongly thanpalmitate. In an alternative embodiment, the hairpin loop adaptor/one ormore second anchors couple (or bind) to the membrane using a mono-acylspecies, such as palmitate, and the Y adaptor/one or more first anchorscouple (or bind) to the membrane using a diacyl species, such asdipalmitoylphosphatidylcholine. Other factors which may affect theability of the one or more groups to couple (or bind) to the membraneinclude, but are not limited to, the charge or hydrophobicity of the oneor more anchors or one or more linkers or the entropic cost. Forinstance, the one or more first and second anchors may comprisecholesterol, but the charge of the one or more second anchors mayincrease the ability of their cholesterol to couple (or bind) to themembrane or the charge of the one or more first anchors may decrease theability of their cholesterol to couple (or bind) to the membrane.

The one or more first anchors may comprise one or more rigid linkers andthe one or more second anchors may comprise one or more flexiblelinkers. Flexible linkers may allow the one or more groups which couple(or bind) to the membrane to move relative to the target double strandedpolynucleotide and thereby increase the likelihood of coupling. In thisembodiment, the one or more groups which couple (or bind) to themembrane may be the same in the first and second anchors. For instance,the one or more first anchors may comprise one or more rigid linkers andcholesterol and the one or more second anchors may comprise one or moreflexible linkers and cholesterol. Suitable linkers are discussed above.Rigid linkers are those which do not substantially bend or flex and donot allow substantial rotation either along their length or at theirattachments points. Rigid linkers are those which do not permitsubstantial movement of the one or more groups which couple (or bind) tothe membrane relative to the target double stranded polynucleotide. Inthe case where a rigid linker comprises a polymeric or oligomericsection, the polymeric or oligomeric section is shorter than thepersistence length, a measure of rigidity, of the corresponding polymeror oligomer. Persistence length can be measured or calculated usingmethods known in the art. For instance, the persistence length ofdouble-stranded DNA is approximately 50 nm or 500 Angstroms or 147nucleotide pairs. A stretch of double-stranded DNA of DNA less than 147nucleotide pairs in length is considered to be rigid. Rigid linkersinclude, but are not limited to, those comprising double strandedpolynucleotides, including DNA, conjugated organic moieties or organicmoieties with bulky side chains that restrict conformational freedom.The one or more rigid linkers preferably comprise one or more doublestranded polynucleotides. Any of the polynucleotides discussed above maybe used. The preferred length of the one or more rigid linkers is 5, 10,15, 20, 25, 27, 30, 35, 40 or more nucleotide pairs.

Flexible linkers are those which substantially bend or flex or rotate.Flexible linkers may bend or flex along their length or at one or bothof their attachment points. Flexible linkers permit substantial movementof the one or more groups which couple (or bind) to the membranerelative to the target double stranded polynucleotide. Flexible linkersare those which permit substantial variation of the orientation of thetarget double stranded polynucleotide. Flexible linkers may allowmovement of the one or more groups by flexing in one or two dimensions.Alternatively or in addition, flexible linkers may allow movement of theone or more groups that is a rotation about an axis. In the case where aflexible linker comprises a polymeric or oligomeric section, thepolymeric or oligomeric section contributes significant flexibility ifit is similar to or longer than the persistence length of thecorresponding polymer or oligomer. Flexible linkers include, but are notlimited to, those comprising a single-stranded oligonucleotide orpolynucleotide, a short carbon spacer (e.g. alkanes and alkenes), apolypeptide, a polyhistidine tag, nucleoside derivatives, a shortpolysaccharide or combinations thereof. Flexible linkers typically alsoallow rotation of the one or more groups which couple (or bind) to themembrane. Flexible linkers may permit rotation in at least one axis. Theaxis is preferably the longitudinal axis of the linker. The one or moreflexible linkers preferably comprise one or more spacer 9 (iSp9) groupsor one or more spacer 18 (iSp18) groups. The one or more flexiblelinkers preferably comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more spacer9 (iSp9) groups and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more spacer 18(iSp18) groups. Preferably (a) the hairpin loop adaptor comprises moreanchors than the Y adaptor, (b) the strength of coupling of one or moresecond anchors to the membrane is greater than the strength of couplingof the one or more first anchors to the membrane, (c) the strength ofcoupling of the one or more second anchors to the hairpin loop adaptoris greater than the strength of coupling of the one or more firstanchors to the Y adaptor, (d) the one or more first anchors and one ormore second anchors couple their respective adaptors to the membrane viahybridisation and the strength of hybridisation is greater in the one ormore second anchors than in the one or more first anchors, (e) the oneor more first anchors comprise one or more rigid linkers and the one ormore second anchors comprise one or more flexible linkers or (f) anycombination of (a) to (e). In other words, the invention preferablycomprises a combination of (a) and (b), a combination of (b) and (c), acombination of (c) and (d), a combination (a) and (c), a combination of(b) and (d), a combination of (a) and (d), a combination of (a) and (e),a combination of (b) and (e), a combination of (c) and (e), acombination of (d) and (e), a combination of (a), (b) and (c), acombination of (b), (c) and (d), a combination of (a), (b) and (d), acombination of (a), (c) and (d), a combination of (a), (b) and (e), acombination of (a), (c) and (e), a combination of (a), (d) and (e), acombination of (b), (c) and (e), a combination of (b), (d) and (e), acombination of (c), (d) and (e), a combination of (a), (b), (c) and (d),a combination of (a), (b), (c) and (e), a combination of (a), (b), (d)and (e), a combination of (a), (c), (d) and (e), a combination of (b),(c), (d) and (e) or a combination of (a), (b), (c), (d) and (e).

Transmembrane Pore

The method comprises taking one or more measurements as at least onestrand of the polynucleotide moves with respect to the transmembranepore. A variety of different types of measurements may be made using thepore. This includes without limitation: electrical measurements andoptical measurements. Possible electrical measurements include: currentmeasurements, impedance measurements, tunnelling measurements (Ivanov APet al., Nano Lett. 2011 Jan. 12;11(1): 279-85), and FET measurements(International Application WO 2005/124888). Optical measurements may becombined with electrical measurements (Soni GV et al., Rev Sci Instrum.2010 January;81(1): 014301). The measurement may be a transmembranecurrent measurement such as measurement of ionic current flowing throughthe pore.

Electrical measurements may be made using standard single channelrecording equipment as describe in Stoddart D et al., Proc Natl AcadSci, 12;106(19): 7702-7, Lieberman KR et al, J Am Chem Soc.2010;132(50): 17961-72, and International Application WO 2000/28312.Alternatively, electrical measurements may be made using a multi-channelsystem, for example as described in International Application WO2009/077734 and International Application WO 2011/067559.

The method is preferably carried out with a potential applied across themembrane. The applied potential may be a voltage potential.Alternatively, the applied potential may be a chemical potential. Anexample of this is using a salt gradient across a membrane, such as anamphiphilic layer. A salt gradient is disclosed in Holden et al., J AmChem Soc. 2007 Jul. 11; 129(27): 8650-5. In some instances, the currentpassing through the detector (or pore) as a polynucleotide moves withrespect to the pore is used to estimate or determine the sequence of thepolynucleotide. This is strand sequencing.

The method comprises contacting the polynucleotide with a transmembranepore. A transmembrane pore is a structure that crosses the membrane tosome degree. It permits hydrated ions driven by an applied potential toflow across or within the membrane. The transmembrane pore typicallycrosses the entire membrane so that hydrated ions may flow from one sideof the membrane to the other side of the membrane. However, thetransmembrane pore does not have to cross the membrane. It may be closedat one end. For instance, the pore may be a well, gap, channel, trenchor slit in the membrane along which or into which hydrated ions mayflow.

Step (c) preferably comprises taking one or more measurements of thecurrent passing through the pore as the at least one strand moves withrespect to the pore wherein the one or more current measurements areindicative of one or more characteristics of the at least one strand.

Any transmembrane pore may be used in the invention. The pore may bebiological or artificial. Suitable pores include, but are not limitedto, protein pores, polynucleotide pores and solid state pores. The poremay be a DNA origami pore (Langecker et al., Science, 2012; 338:932-936).

The transmembrane pore is preferably a transmembrane protein pore. Atransmembrane protein pore is a polypeptide or a collection ofpolypeptides that permits hydrated ions, such as an analyte, to flowfrom one side of a membrane to the other side of the membrane. In thepresent invention, the transmembrane protein pore is capable of forminga pore that permits hydrated ions driven by an applied potential to flowfrom one side of the membrane to the other. The transmembrane proteinpore preferably permits analyte such as nucleotides to flow from oneside of the membrane, such as a triblock copolymer membrane, to theother. The transmembrane protein pore allows a polynucleotide, such asDNA or RNA, to be moved through the pore.

The transmembrane protein pore may be a monomer or an oligomer. The poreis preferably made up of several repeating subunits, such as at least 3,at least 4, at least 5, at least 6, at least 7, at least 8, at least 9,at least 10, at least 11, at least 12, at least 13 or at least 14subunits, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 subunits.The pore is preferably a hexameric, heptameric, octameric or nonamericpore. The pore may be a homo-oligomer or a hetero-oligomer.

The transmembrane protein pore typically comprises a barrel or channelthrough which the ions may flow. The subunits of the pore typicallysurround a central axis and contribute strands to a transmembrane βbarrel or channel or a transmembrane α-helix bundle or channel.

The barrel or channel of the transmembrane protein pore typicallycomprises amino acids that facilitate interaction with analyte, such asnucleotides, polynucleotides or nucleic acids. These amino acids arepreferably located near a constriction of the barrel or channel. Thetransmembrane protein pore typically comprises one or more positivelycharged amino acids, such as arginine, lysine or histidine, or aromaticamino acids, such as tyrosine or tryptophan. These amino acids typicallyfacilitate the interaction between the pore and nucleotides,polynucleotides or nucleic acids.

Transmembrane protein pores for use in accordance with the invention canbe derived from β-barrel pores or α-helix bundle pores. β-barrel porescomprise a barrel or channel that is formed from β-strands. Suitableβ-barrel pores include, but are not limited to, β-toxins, such asα-hemolysin, anthrax toxin and leukocidins, and outer membraneproteins/porins of bacteria, such as Mycobacterium smegmatis porin(Msp), for example MspA, MspB, MspC or MspD, lysenin, outer membraneporin F (OmpF), outer membrane porin G (OmpG), outer membranephospholipase A and Neisseria autotransporter lipoprotein (NalP).α-helix bundle pores comprise a barrel or channel that is formed fromα-helices. Suitable α-helix bundle pores include, but are not limitedto, inner membrane proteins and α outer membrane proteins, such as WZAand ClyA toxin. The transmembrane pore may be derived from lysenin.Suitable pores derived from lysenin are disclosed in InternationalApplication No. PCT/GB2013/050667 (published as WO 2013/153359). Thetransmembrane pore may be derived from Msp or from α-hemolysin (α-HL).

The transmembrane protein pore is preferably derived from Msp,preferably from MspA. Such a pore will be oligomeric and typicallycomprises 7, 8, 9 or 10 monomers derived from Msp. The pore may be ahomo-oligomeric pore derived from Msp comprising identical monomers.Alternatively, the pore may be a hetero-oligomeric pore derived from Mspcomprising at least one monomer that differs from the others. Preferablythe pore is derived from MspA or a homolog or paralog thereof.

A monomer derived from Msp typically comprises the sequence shown in SEQID NO: 2 or a variant thereof. SEQ ID NO: 2 is the MS-(B1)8 mutant ofthe MspA monomer. It includes the following mutations: D90N, D91N, D93N,D118R, D134R and E139K. A variant of SEQ ID NO: 2 is a polypeptide thathas an amino acid sequence which varies from that of SEQ ID NO: 2 andwhich retains its ability to form a pore. The ability of a variant toform a pore can be assayed using any method known in the art. Forinstance, the variant may be inserted into an amphiphilic layer alongwith other appropriate subunits and its ability to oligomerise to form apore may be determined. Methods are known in the art for insertingsubunits into membranes, such as amphiphilic layers. For example,subunits may be suspended in a purified form in a solution containing atriblock copolymer membrane such that it diffuses to the membrane and isinserted by binding to the membrane and assembling into a functionalstate. Alternatively, subunits may be directly inserted into themembrane using the “pick and place” method described in M. A. Holden, H.Bayley. J. Am. Chem. Soc. 2005, 127, 6502-6503 and InternationalApplication No. PCT/GB2006/001057 (published as WO 2006/100484).

Over the entire length of the amino acid sequence of SEQ ID NO: 2, avariant will preferably be at least 50% homologous to that sequencebased on amino acid identity. More preferably, the variant may be atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90% and more preferably at least 95%,97% or 99% homologous based on amino acid identity to the amino acidsequence of SEQ ID NO: 2 over the entire sequence. There may be at least80%, for example at least 85%, 90% or 95%, amino acid identity over astretch of 100 or more, for example 125, 150, 175 or 200 or more,contiguous amino acids (“hard homology”).

Standard methods in the art may be used to determine homology. Forexample the UWGCG Package provides the BESTFIT program which can be usedto calculate homology, for example used on its default settings(Devereux et al (1984) Nucleic Acids Research 12, p387-395). The PILEUPand BLAST algorithms can be used to calculate homology or line upsequences (such as identifying equivalent residues or correspondingsequences (typically on their default settings)), for example asdescribed in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S. Fet al (1990) J Mol Biol 215: 403-10. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/).

SEQ ID NO: 2 is the MS-(B1)8 mutant of the MspA monomer. The variant maycomprise any of the mutations in the MspB, C or D monomers compared withMspA. The mature forms of MspB, C and D are shown in SEQ ID NOs: 5 to 7.In particular, the variant may comprise the following substitutionpresent in MspB: A138P. The variant may comprise one or more of thefollowing substitutions present in MspC: A96G, N102E and A138P. Thevariant may comprise one or more of the following mutations present inMspD: Deletion of G1, L2V, E5Q, L8V, DβG, W21A, D22E, K47T, I49H, I68V,D91G, A96Q, N102D, S103T, V104I, S136K and G141A. The variant maycomprise combinations of one or more of the mutations and substitutionsfrom Msp B, C and D. The variant preferably comprises the mutation L88N.A variant of SEQ ID NO: 2 has the mutation L88N in addition to all themutations of MS-B1 and is called MS-(B2)8. The pore used in theinvention is preferably MS-(B2)8. A variant of SEQ ID NO: 2 has themutations G75S/G77S/L88N/Q126R in addition to all the mutations of MS-B1and is called MS-B2C. The pore used in the invention is preferablyMS-(B2)8 or MS-(B2C)8.

Amino acid substitutions may be made to the amino acid sequence of SEQID NO: 2 in addition to those discussed above, for example up to 1, 2,3, 4, 5, 10, 20 or 30 substitutions. Conservative substitutions replaceamino acids with other amino acids of similar chemical structure,similar chemical properties or similar side-chain volume. The aminoacids introduced may have similar polarity, hydrophilicity,hydrophobicity, basicity, acidity, neutrality or charge to the aminoacids they replace. Alternatively, the conservative substitution mayintroduce another amino acid that is aromatic or aliphatic in the placeof a pre-existing aromatic or aliphatic amino acid.

One or more amino acid residues of the amino acid sequence of SEQ ID NO:2 may additionally be deleted from the polypeptides described above. Upto 1, 2, 3, 4, 5, 10, 20 or 30 residues may be deleted, or more.

Variants may include fragments of SEQ ID NO: 2. Such fragments retainpore forming activity. Fragments may be at least 50, 100, 150 or 200amino acids in length. Such fragments may be used to produce the pores.A fragment preferably comprises the pore forming domain of SEQ ID NO: 2.Fragments must include one of residues 88, 90, 91, 105, 118 and 134 ofSEQ ID NO: 2. Typically, fragments include all of residues 88, 90, 91,105, 118 and 134 of SEQ ID NO: 2.

One or more amino acids may be alternatively or additionally added tothe polypeptides described above. An extension may be provided at theamino terminal or carboxy terminal of the amino acid sequence of SEQ IDNO: 2 or polypeptide variant or fragment thereof. The extension may bequite short, for example from 1 to 10 amino acids in length.Alternatively, the extension may be longer, for example up to 50 or 100amino acids. A carrier protein may be fused to an amino acid sequenceaccording to the invention. Other fusion proteins are discussed in moredetail below.

As discussed above, a variant is a polypeptide that has an amino acidsequence which varies from that of SEQ ID NO: 2 and which retains itsability to form a pore. A variant typically contains the regions of SEQID NO: 2 that are responsible for pore formation. The pore formingability of Msp, which contains a β-barrel, is provided by β-sheets ineach subunit. A variant of SEQ ID NO: 2 typically comprises the regionsin SEQ ID NO: 2 that form β-sheets. One or more modifications can bemade to the regions of SEQ ID NO: 2 that form β-sheets as long as theresulting variant retains its ability to form a pore. A variant of SEQID NO: 2 preferably includes one or more modifications, such assubstitutions, additions or deletions, within its a-helices and/or loopregions.

The monomer derived from Msp contains one or more specific modificationsto facilitate nucleotide discrimination. The monomer derived from Mspmay also contain other non-specific modifications as long as they do notinterfere with pore formation. A number of non-specific side chainmodifications are known in the art and may be made to the side chains ofthe monomer derived from Msp. Such modifications include, for example,reductive alkylation of amino acids by reaction with an aldehydefollowed by reduction with NaBH_(4,) amidination with methylacetimidateor acylation with acetic anhydride.

The monomer derived from Msp can be produced using standard methodsknown in the art. The monomer derived from Msp may be made syntheticallyor by recombinant means. For example, the pore may be synthesized by invitro translation and transcription (IVTT). Suitable methods forproducing pores are discussed in International Application Nos.PCT/GB09/001690 (published as WO 2010/004273), PCT/GB09/001679(published as WO 2010/004265) or PCT/GB10/000133 (published as WO2010/086603). Methods for inserting pores into membranes are discussed.

The transmembrane protein pore is also preferably derived fromα-hemolysin (α-HL). The wild type α-HL pore is formed of seven identicalmonomers or subunits (i.e. it is heptameric). The sequence of onemonomer or subunit of α-hemolysin-NN is shown in SEQ ID NO: 4.

In some embodiments, the transmembrane protein pore is chemicallymodified. The pore can be chemically modified in any way and at anysite. The transmembrane protein pore is preferably chemically modifiedby attachment of a molecule to one or more cysteines (cysteine linkage),attachment of a molecule to one or more lysines, attachment of amolecule to one or more non-natural amino acids, enzyme modification ofan epitope or modification of a terminus. Suitable methods for carryingout such modifications are well-known in the art. The transmembraneprotein pore may be chemically modified by the attachment of anymolecule. For instance, the pore may be chemically modified byattachment of a dye or a fluorophore.

Any number of the monomers in the pore may be chemically modified. Oneor more, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10, of the monomers ispreferably chemically modified as discussed above.

The reactivity of cysteine residues may be enhanced by modification ofthe adjacent residues. For instance, the basic groups of flankingarginine, histidine or lysine residues will change the pKa of thecysteines thiol group to that of the more reactive S⁻ group. Thereactivity of cysteine residues may be protected by thiol protectivegroups such as dTNB. These may be reacted with one or more cysteineresidues of the pore before a linker is attached.

The molecule (with which the pore is chemically modified) may beattached directly to the pore or attached via a linker as disclosed inInternational Application Nos. PCT/GB09/001690 (published as WO2010/004273), PCT/GB09/001679 (published as WO 2010/004265) orPCT/GB10/000133 (published as WO 2010/086603).

Any of the proteins described herein, such as the transmembrane proteinpores, can be produced using standard methods known in the art.Polynucleotide sequences encoding a pore or construct may be derived andreplicated using standard methods in the art. Polynucleotide sequencesencoding a pore or construct may be expressed in a bacterial host cellusing standard techniques in the art. The pore may be produced in a cellby in situ expression of the polypeptide from a recombinant expressionvector. The expression vector optionally carries an inducible promoterto control the expression of the polypeptide. These methods aredescribed in Sambrook, J. and Russell, D. (2001). Molecular Cloning: ALaboratory Manual, 3rd Edition. Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.

The pore may be produced in large scale following purification by anyprotein liquid chromatography system from protein producing organisms orafter recombinant expression. Typical protein liquid chromatographysystems include FPLC, AKTA systems, the Bio-Cad system, the Bio-RadBioLogic system and the Gilson HPLC system.

Polynucleotide Characterisation

The method of the invention involves measuring one or morecharacteristics of the target double stranded polynucleotide.

Any number of polynucleotides can be investigated. For instance, themethod of the invention may concern characterising 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 50, 100 or more polynucleotides.

The polynucleotides can be naturally occurring or artificial. Forinstance, the method may be used to verify the sequence of two or moremanufactured oligonucleotides. The methods are typically carried out invitro.

The method may involve measuring two, three, four or five or morecharacteristics of the polynucleotide. The one or more characteristicsare preferably selected from (i) the length of the polynucleotide, (ii)the identity of the polynucleotide, (iii) the sequence of thepolynucleotide, (iv) the secondary structure of the polynucleotide and(v) whether or not the polynucleotide is modified. Any combination of(i) to (v) may be measured in accordance with the invention, such as{i}, {ii}, {iii}, {iv}, {v}, {i,ii}, {i,iii}, {i,iv}, {i,v}, {ii,iii},{ii,iv}, {ii,v}, {iii,iv}, {iii,v}, {iv,v}, {i,ii,iii}, {i,ii,iv},{i,ii,v}, {i,iii,iv}, {i,iii,v}, {i,iv,v}, {ii,iii,iv}, {ii,iii,v},{ii,iv,v}, {iii,iv,v}, {i,ii,iii,iv}, {i,ii,iii,v}, {i,ii,iv,v},{i,iii,iv,v}, {ii,iii,iv,v} or {i,ii,iii,iv,v}. Different combinationsof (i) to (v) may be measured for the first polynucleotide compared withthe second polynucleotide, including any of those combinations listedabove.

For (i), the length of the polynucleotide may be measured for example bydetermining the number of interactions between the polynucleotide andthe pore or the duration of interaction between the polynucleotide andthe pore.

For (ii), the identity of the polynucleotide may be measured in a numberof ways. The identity of the polynucleotide may be measured inconjunction with measurement of the sequence of the polynucleotide orwithout measurement of the sequence of the polynucleotide. The former isstraightforward; the polynucleotide is sequenced and thereby identified.The latter may be done in several ways. For instance, the presence of aparticular motif in the polynucleotide may be measured (withoutmeasuring the remaining sequence of the polynucleotide). Alternatively,the measurement of a particular electrical and/or optical signal in themethod may identify the polynucleotide as coming from a particularsource.

For (iii), the sequence of the polynucleotide can be determined asdescribed previously. Suitable sequencing methods, particularly thoseusing electrical measurements, are described in Stoddart D et al., ProcNatl Acad Sci, 12;106(19): 7702-7, Lieberman K R et al, J Am Chem Soc.2010;132(50): 17961-72, and International Application WO 2000/28312.

For (iv), the secondary structure may be measured in a variety of ways.For instance, if the method involves an electrical measurement, thesecondary structure may be measured using a change in dwell time or achange in current flowing through the pore. This allows regions ofsingle-stranded and double-stranded polynucleotide to be distinguished.

For (v), the presence or absence of any modification may be measured.The method preferably comprises determining whether or not thepolynucleotide is modified by methylation, by oxidation, by damage, withone or more proteins or with one or more labels, tags or spacers.Specific modifications will result in specific interactions with thepore which can be measured using the methods described below. Forinstance, methylcytosine may be distinguished from cytosine on the basisof the current flowing through the pore during its interaction with eachnucleotide.

The methods may be carried out using any apparatus that is suitable forinvestigating a membrane/pore system in which a pore is present in amembrane. The method may be carried out using any apparatus that issuitable for transmembrane pore sensing. For example, the apparatuscomprises a chamber comprising an aqueous solution and a barrier thatseparates the chamber into two sections. The barrier typically has anaperture in which the membrane containing the pore is formed.Alternatively the barrier forms the membrane in which the pore ispresent.

The methods may be carried out using the apparatus described inInternational Application No. PCT/GB08/000562 (WO 2008/102120).

The methods may involve measuring the current passing through the poreas the polynucleotide moves with respect to the pore. Therefore theapparatus may also comprise an electrical circuit capable of applying apotential and measuring an electrical signal across the membrane andpore. The methods may be carried out using a patch clamp or a voltageclamp. The methods preferably involve the use of a voltage clamp.

The methods of the invention may involve the measuring of a currentpassing through the pore as the polynucleotide moves with respect to thepore. Suitable conditions for measuring ionic currents throughtransmembrane protein pores are known in the art and disclosed in theExample. The method is typically carried out with a voltage appliedacross the membrane and pore. The voltage used is typically from +5 V to−5 V, such as from +4 V to -4 V, +3 V to −3 V or +2 V to −2 V. Thevoltage used is typically from −600 mV to +600 mV or −400 mV to +400 mV.The voltage used is preferably in a range having a lower limit selectedfrom −400 mV, −300 mV, −200 mV, −150 mV, −100 mV, −50 mV, −20mV and 0 mVand an upper limit independently selected from +10 mV, +20 mV, +50 mV,+100 mV, +150 mV, +200 mV, +300 mV and +400 mV. The voltage used is morepreferably in the range 100 mV to 240 mV and most preferably in therange of 120 mV to 220 mV. It is possible to increase discriminationbetween different nucleotides by a pore by using an increased appliedpotential.

The methods are typically carried out in the presence of any chargecarriers, such as metal salts, for example alkali metal salt, halidesalts, for example chloride salts, such as alkali metal chloride salt.Charge carriers may include ionic liquids or organic salts, for exampletetramethyl ammonium chloride, trimethylphenyl ammonium chloride,phenyltrimethyl ammonium chloride, or 1-ethyl-3-methyl imidazoliumchloride. In the exemplary apparatus discussed above, the salt ispresent in the aqueous solution in the chamber. Potassium chloride(KCl), sodium chloride (NaCl), caesium chloride (CsCl) or a mixture ofpotassium ferrocyanide and potassium ferricyanide is typically used.KCl, NaCl and a mixture of potassium ferrocyanide and potassiumferricyanide are preferred. The charge carriers may be asymmetric acacross the membrane. The type and/or concentration of the chargecarriers may be different on each side of the membrane.

The salt concentration may be at saturation. The salt concentration maybe 3 M or lower and is typically from 0.1 to 2.5 M, from 0.3 to 1.9 M,from 0.5 to 1.8 M, from 0.7 to 1.7 M, from 0.9 to 1.6 M or from 1 M to1.4 M. The salt concentration is preferably from 150 mM to 1 M. Themethod is preferably carried out using a salt concentration of at least0.3 M, such as at least 0.4 M, at least 0.5 M, at least 0.6 M, at least0.8 M, at least 1.0 M, at least 1.5 M, at least 2.0 M, at least 2.5 M orat least 3.0 M. High salt concentrations provide a high signal to noiseratio and allow for currents indicative of the presence of a nucleotideto be identified against the background of normal current fluctuations.

The methods are typically carried out in the presence of a buffer. Inthe exemplary apparatus discussed above, the buffer is present in theaqueous solution in the chamber. Any buffer may be used in the method ofthe invention. Typically, the buffer is phosphate buffer. Other suitablebuffers are HEPES and Tris-HCl buffer. The methods are typically carriedout at a pH of from 4.0 to 12.0, from 4.5 to 10.0, from 5.0 to 9.0, from5.5 to 8.8, from 6.0 to 8.7 or from 7.0 to 8.8 or 7.5 to 8.5. The pHused is preferably about 7.5.

The methods may be carried out at from 0° C. to 100° C., from 15° C. to95° C., from 16° C. to 90° C., from 17° C. to 85° C., from 18° C. to 80°C., 19° C. to 70° C., or from 20° C. to 60° C. The methods are typicallycarried out at room temperature. The methods are optionally carried outat a temperature that supports enzyme function, such as about 37° C.

Step (b) preferably comprises contacting the polynucleotide provided instep a) with a polynucleotide binding protein such that the proteincontrols the movement of the at least one strand of the polynucleotidethrough the pore.

More preferably, the method comprises b) contacting the polynucleotideprovided in step a) with a transmembrane pore and a polynucleotidebinding protein such that at least one strand of the polynucleotidemoves through the pore and the protein controls the movement of the atleast one strand of the polynucleotide through the pore; and c)measuring the current passing through the pore as the at least onestrand of the polynucleotide moves with respect to the pore wherein thecurrent is indicative of one or more characteristics of the at least onestrand of the polynucleotide and thereby characterising the doublestranded target polynucleotide.

The polynucleotide binding protein may be any protein that is capable ofbinding to the polynucleotide and controlling its movement through thepore. It is straightforward in the art to determine whether or not aprotein binds to a polynucleotide. The protein typically interacts withand modifies at least one property of the polynucleotide. The proteinmay modify the polynucleotide by cleaving it to form individualnucleotides or shorter chains of nucleotides, such as di- ortrinucleotides. The moiety may modify the polynucleotide by orienting itor moving it to a specific position, i.e. controlling its movement.

The polynucleotide binding protein is preferably derived from apolynucleotide handling enzyme. A polynucleotide handling enzyme is apolypeptide that is capable of interacting with and modifying at leastone property of a polynucleotide. The enzyme may modify thepolynucleotide by cleaving it to form individual nucleotides or shorterchains of nucleotides, such as di- or trinucleotides. The enzyme maymodify the polynucleotide by orienting it or moving it to a specificposition. The polynucleotide handling enzyme does not need to displayenzymatic activity as long as it is capable of binding thepolynucleotide and controlling its movement through the pore. Forinstance, the enzyme may be modified to remove its enzymatic activity ormay be used under conditions which prevent it from acting as an enzyme.Such conditions are discussed in more detail below.

The polynucleotide handling enzyme is preferably derived from anucleolytic enzyme. The polynucleotide handling enzyme used is morepreferably derived from a member of any of the Enzyme Classification(EC) groups 3.1.11, 3.1.13, 3.1.14, 3.1.15, 3.1.16, 3.1.21, 3.1.22,3.1.25, 3.1.26, 3.1.27, 3.1.30 and 3.1.31. The enzyme may be any ofthose disclosed in International Application No. PCT/GB10/000133(published as WO 2010/086603).

Preferred enzymes are polymerases, exonucleases, helicases andtopoisomerases, such as gyrases. Suitable enzymes include, but are notlimited to, exonuclease I from E. coli (SEQ ID NO: 11), exonuclease IIIenzyme from E. coli (SEQ ID NO: β), RecJ from T. thermophilus (SEQ IDNO: 15) and bacteriophage lambda exonuclease (SEQ ID NO: 17) andvariants thereof. Three subunits comprising the sequence shown in SEQ IDNO: 15 or a variant thereof interact to form a trimer exonuclease. Theenzyme is preferably Phi29 DNA polymerase (SEQ ID NO: 9) or a variantthereof. The topoisomerase is preferably a member of any of the MoietyClassification (EC) groups 5.99.1.2 and 5.99.1.3.

The enzyme is most preferably derived from a helicase, such as He1308Mbu (SEQ ID NO: 18), Hel308 Csy (SEQ ID NO: 19), Hel308 Mhu (SEQ ID NO:21), Tral Eco (SEQ ID NO: 22), XPD Mbu (SEQ ID NO: 23) or a variantthereof. Any helicase may be used in the invention. The helicase may beor be derived from a Hel308 helicase, a RecD helicase, such as Tralhelicase or a TrwC helicase, a XPD helicase or a Dda helicase. Thehelicase may be any of the helicases, modified helicases or helicaseconstructs disclosed in International Application Nos. PCT/GB2012/052579(published as WO 2013/057495); PCT/GB2012/053274 (published as WO2013/098562); PCT/GB2012/053273 (published as WO2013098561);PCT/GB2013/051925 (published as WO 2014/013260); PCT/GB2013/051924(published as WO 2014/013259); PCT/GB2013/051928 (published as WO2014/013262) and PCT/GB2014/052736.

The helicase preferably comprises the sequence shown in SEQ ID NO: 25(Trwc Cba) or as variant thereof, the sequence shown in SEQ ID NO: 18(Hel308 Mbu) or a variant thereof or the sequence shown in SEQ ID NO: 24(Dda) or a variant thereof. Variants may differ from the nativesequences in any of the ways discussed below for transmembrane pores. Apreferred variant of SEQ ID NO: 24 comprises E94C/A360C and then(ΔM1)G1G2 (i.e. deletion of M1 and then addition G1 and G2) orE94C/A360C/C109A/C136A and then (ΔM1)G1G2.

Any number of helicases may be used in accordance with the invention.For instance, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more helicases may beused. In some embodiments, different numbers of helicases may be used.

The method of the invention preferably comprises contacting the targetdouble stranded polynucleotide with two or more helicases. The two ormore helicases are typically the same helicase. The two or morehelicases may be different helicases.

The two or more helicases may be any combination of the helicasesmentioned above. The two or more helicases may be two or more Ddahelicases. The two or more helicases may be one or more Dda helicasesand one or more TrwC helicases. The two or more helicases may bedifferent variants of the same helicase.

The two or more helicases are preferably attached to one another. Thetwo or more helicases are more preferably covalently attached to oneanother. The helicases may be attached in any order and using anymethod. Preferred helicase constructs for use in the invention aredescribed in International Application Nos. PCT/GB2013/051925 (publishedas WO 2014/013260); PCT/GB2013/051924 (published as WO 2014/013259);PCT/GB2013/051928 (published as WO 2014/013262) and PCT/GB2014/052736.

A variant of SEQ ID NOs: 9, 11, 13, 15, 17, 18, 19, 20, 21, 22, 23, 24or 25 is an enzyme that has an amino acid sequence which varies fromthat of SEQ ID NO: 9, 11, 13, 15, 17, 18, 19, 20, 21, 22, 23, 24 or 25and which retains polynucleotide binding ability. This can be measuredusing any method known in the art. For instance, the variant can becontacted with a polynucleotide and its ability to bind to and movealong the polynucleotide can be measured. The variant may includemodifications that facilitate binding of the polynucleotide and/orfacilitate its activity at high salt concentrations and/or roomtemperature. Variants may be modified such that they bindpolynucleotides (i.e. retain polynucleotide binding ability) but do notfunction as a helicase (i.e. do not move along polynucleotides whenprovided with all the necessary components to facilitate movement, e.g.ATP and Mg²⁺). Such modifications are known in the art. For instance,modification of the Mg²⁺ binding domain in helicases typically resultsin variants which do not function as helicases. These types of variantsmay act as molecular brakes.

Over the entire length of the amino acid sequence of SEQ ID NO: 9, 11,13, 15, 17, 18, 19, 20, 21, 22, 23, 24 or 25, a variant will preferablybe at least 50% homologous to that sequence based on amino acididentity. More preferably, the variant polypeptide may be at least 55%,at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90% and more preferably at least 95%, 97% or 99%homologous based on amino acid identity to the amino acid sequence ofSEQ ID NO: 9, 11, 13, 15, 17, 18, 19, 20, 21, 22, 23, 24 or 25 over theentire sequence. There may be at least 80%, for example at least 85%,90% or 95%, amino acid identity over a stretch of 200 or more, forexample 230, 250, 270, 280, 300, 400, 500, 600, 700, 800, 900 or 1000 ormore, contiguous amino acids (“hard homology”). Homology is determinedas described above. The variant may differ from the wild-type sequencein any of the ways discussed above with reference to SEQ ID NO: 2 and 4above. The enzyme may be covalently attached to the pore. Any method maybe used to covalently attach the enzyme to the pore.

A preferred molecular brake is TrwC Cba-Q594A (SEQ ID NO: 25 with themutation Q594A). This variant does not function as a helicase (i.e.binds polynucleotides but does not move along them when provided withall the necessary components to facilitate movement, e.g. ATP and Mg²⁻).

In strand sequencing, the polynucleotide is translocated through thepore either with or against an applied potential. Exonucleases that actprogressively or processively on double stranded polynucleotides can beused on the cis side of the pore to feed the remaining single strandthrough under an applied potential or the trans side under a reversepotential. Likewise, a helicase that unwinds the double stranded DNA canalso be used in a similar manner. A polymerase may also be used. Thereare also possibilities for sequencing applications that require strandtranslocation against an applied potential, but the DNA must be first“caught” by the enzyme under a reverse or no potential. With thepotential then switched back following binding the strand will pass cisto trans through the pore and be held in an extended conformation by thecurrent flow. The single strand DNA exonucleases or single strand DNAdependent polymerases can act as molecular motors to pull the recentlytranslocated single strand back through the pore in a controlledstepwise manner, trans to cis, against the applied potential.

Helicase(s) and Molecular Brake(s)

In a preferred embodiment, the invention provides a method ofcontrolling the movement of a double stranded polynucleotide through atransmembrane pore, comprising:

(a) providing the double stranded polynucleotide with a Y adaptor at oneend and a hairpin loop adaptor at the other end, wherein the Y adaptorcomprises the one or more helicases and one or more first anchors forcoupling the polynucleotide to the membrane, wherein the hairpin loopadaptor comprises the one or more molecular brakes and one or moresecond anchors for coupling the polynucleotide to the membrane andwherein the strength of coupling of the hairpin loop adaptor to themembrane is greater than the strength of coupling of the Y adaptor tothe membrane;

(b) contacting the target polynucleotide provided in step (a) with thepore; and

(c) applying a potential across the pore such that the one or morehelicases and the one or more molecular brakes are brought together andboth control the movement of the target polynucleotide through the pore.

This type of method is discussed in detail in the InternationalApplication PCT/GB2014/052737.

The one or more molecular brakes are preferably one or morepolynucleotide binding proteins. The polynucleotide binding protein maybe any protein that is capable of binding to the polynucleotide andcontrolling its movement through the pore. It is straightforward in theart to determine whether or not a protein binds to a polynucleotide. Theprotein typically interacts with and modifies at least one property ofthe polynucleotide. The protein may modify the polynucleotide bycleaving it to form individual nucleotides or shorter chains ofnucleotides, such as di- or trinucleotides. The moiety may modify thepolynucleotide by orienting it or moving it to a specific position, i.e.controlling its movement.

The polynucleotide binding protein is preferably derived from apolynucleotide handling enzyme. The one or more molecular brakes may bederived from any of the polynucleotide handling enzymes discussed above.Modified versions of Phi29 polymerase (SEQ ID NO: 8) which act asmolecular brakes are disclosed in U.S. Pat. No. 5,576,204. The one ormore molecular brakes are preferably derived from a helicase.

Spacers in the Y Adaptor

The one or more helicases attached to the Y adaptor may be stalled atone or more spacers as discussed in International Application No.PCT/GB2014/050175 (published as WO 2014/135838). Any configuration ofone or more helicases and one or more spacers disclosed in theInternational Application may be used in this invention.

RTC Sequencing In a preferred embodiment, step b) comprises contactingthe polynucleotide provided in step a) with a transmembrane pore suchthat both strands of the polynucleotide move through the pore and stepc) comprises taking one or more measurements as the both strands of thepolynucleotide move with respect to the pore wherein the measurementsare indicative of one or more characteristics of the strands of thepolynucleotide and thereby characterising the target polynucleotide. Anyof the embodiments discussed above equally apply to this embodiment.

Uncoupling

The method of the invention may involve characterising multiple targetdouble stranded polynucleotides and uncoupling of the first targetdouble stranded polynucleotide.

In a preferred embodiment, the invention involves characterising two ormore target double stranded polynucleotides. The method preferablycomprises:

(a) providing a first target double stranded polynucleotide in a firstsample with a first Y adaptor at one end and a first hairpin loopadaptor at the other end, wherein the first Y adaptor comprises one ormore first anchors for coupling the polynucleotide to the membrane,wherein the first hairpin loop adaptor comprises one or more secondanchors for coupling the polynucleotide to the membrane and wherein thestrength of coupling of the first hairpin loop adaptor to the membraneis greater than the strength of coupling of the first Y adaptor to themembrane;

(b) providing a second target double stranded polynucleotide in a secondsample with a second Y adaptor at one end and a second hairpin loopadaptor at the other end, wherein the second Y adaptor comprises one ormore third anchors for coupling the polynucleotide to the membrane,wherein the second hairpin loop adaptor comprises one or more fourthanchors for coupling the polynucleotide to the membrane and wherein thestrength of coupling of the second hairpin loop adaptor to the membraneis greater than the strength of coupling of the second Y adaptor to themembrane;

(c) coupling the first polynucleotide provided in step (a) to amembrane;

(d) contacting the first polynucleotide coupled in step (c) with atransmembrane pore such that at least one strand of the firstpolynucleotide moves through the pore;

(e) taking one or more measurements as the at least one strand of thefirst polynucleotide moves with respect to the pore wherein themeasurements are indicative of one or more characteristics of the atleast one strand of the first polynucleotide and thereby characterisingthe first polynucleotide;

(f) uncoupling the first polynucleotide from the membrane;

(g) coupling the second polynucleotide provided in step (b) to themembrane;

(h) contacting the second polynucleotide coupled in step (g) with atransmembrane pore such that at least one strand of the secondpolynucleotide moves through the pore; and

(i) taking one or more measurements as the at least one strand of thesecond polynucleotide moves with respect to the pore wherein themeasurements are indicative of one or more characteristics of the atleast one strand of the second polynucleotide and thereby characterisingthe first polynucleotide.

This type of method is discussed in detail in the UK Application1406155.0 and in the International application being filed concurrentlywith this application (ONT IP 055). Any of the embodiments discussedtherein are applicable to this method.

Step (f) (i.e. uncoupling of the first polynucleotide) may be performedbefore step (g) (i.e. before coupling the second polynucleotide to themembrane). Step (g) may be performed before step (f). If the secondpolynucleotide is coupled to the membrane before the firstpolynucleotide is uncoupled, step (f) preferably comprises selectivelyuncoupling the first polynucleotide from the membrane (i.e. uncouplingthe first polynucleotide but not the second polynucleotide from themembrane). A skilled person can design a system in which selectiveuncoupling is achieved. Steps (f) and (g) may be performed at the sametime. This is discussed in more detail below.

Removal or Washing

Although the first polynucleotide is uncoupled from the membrane in step(f), it is not necessarily removed or washed away. If the secondpolynucleotide can be easily distinguished from the firstpolynucleotide, there is no need to remove the first polynucleotide.

Between steps (f) and (g), the method preferably further comprisesremoving at least some of the first sample from the membrane. At least10% of the first sample may be removed, such as at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80% or at least 90% of the first sample may be removed.

The method more preferably further comprises removing all of the firstsample from the membrane. This can be done in any way. For instance, themembrane can be washed with a buffer after the first polynucleotide hasbeen uncoupled. Suitable buffers are discussed below.

Modified Target Double Stranded Polynucleotide

Before step a), a target double stranded polynucleotide may be contactedwith a polymerase and a population of free nucleotides under conditionsin which the polymerase forms a modified target double strandedpolynucleotide using the target double stranded polynucleotide as atemplate, wherein the polymerase replaces one or more of the nucleotidespecies in the target polynucleotide with a different nucleotide specieswhen forming the modified polynucleotide. The modified target doublestranded polynucleotide may then be provided as in step a). This type ofmodification is described in the International Application No.PCT/GB2015/050483. Any of the polymerases discussed above may be used.The polymerase is preferably Klenow or 9° North.

The template polynucleotide is contacted with the polymerase underconditions in which the polymerase forms a modified polynucleotide usingthe template polynucleotide as a template. Such conditions are known inthe art. For instance, the polynucleotide is typically contacted withthe polymerase in commercially available polymerase buffer, such asbuffer from New England Biolabs®. The temperature is preferably from 20to 37° C. for Klenow or from 60 to 75° C. for 9° North. A primer or a 3′hairpin is typically used as the nucleation point for polymeraseextension.

Characterisation, such as sequencing, of a polynucleotide using atransmembrane pore typically involves analyzing polymer units made up ofk nucleotides where k is a positive integer (i.e. ‘k-mers’). This isdiscussed in International Application No. PCT/GB2012/052343 (publishedas WO 2013/041878). While it is desirable to have clear separationbetween current measurements for different k-mers, it is common for someof these measurements to overlap. Especially with high numbers ofpolymer units in the k-mer, i.e. high values of k, it can becomedifficult to resolve the measurements produced by different k-mers, tothe detriment of deriving information about the polynucleotide, forexample an estimate of the underlying sequence of the polynucleotide.

By replacing one or more nucleotide species in the target polynucleotidewith different nucleotide species in the modified nucleotide, themodified polynucleotide contains k-mers which differ from those in thetarget polynucleotide. The different k-mers in the modifiedpolynucleotide are capable of producing different current measurementsfrom the k-mers in the target polynucleotide and so the modifiedpolynucleotide provides different information from the targetpolynucleotide. The additional information from the modifiedpolynucleotide can make it easier to characterise the targetpolynucleotide. In some instances, the modified polynucleotide itselfmay be easier to characterise. For instance, the modified polynucleotidemay be designed to include k-mers with an increased separation or aclear separation between their current measurements or k-mers which havea decreased noise.

Other Characterisation Method

In another embodiment, the polynucleotide is characterised by detectinglabelled species that are released as a polymerase incorporatesnucleotides into the polynucleotide. The polymerase uses thepolynucleotide as a target. Each labelled species is specific for eachnucleotide. In step a) the target double stranded polynucleotide isprovided as discussed above. In step b), the polynucleotide provided instep a) is contacted with the transmembrane pore, a polymerase andlabelled nucleotides such that phosphate labelled species aresequentially released when nucleotides are added to the polynucleotideby the polymerase, wherein the phosphate species contain a labelspecific for each nucleotide. The polymerase may be any of thosediscussed above. In step c), the phosphate labelled species are detectedusing the pore and thereby characterising the double stranded targetpolynucleotide. Steps b) and c) are disclosed in European ApplicationNo. 13187149.3 (published as EP 2682460). Any of the embodimentsdiscussed above equally apply to this method.

Modification Methods of the Invention

The invention also provides methods of producing the modified targetpolynucleotide provided in step a) of the characterisation method.

The invention provides a method for modifying a target double strandedpolynucleotide for characterisation using a transmembrane pore in amembrane. The method involves ligating a Y adaptor to one end of thepolynucleotide and ligating a hairpin loop adaptor to the other end ofthe polynucleotide. As above, the Y adaptor comprises one or more firstanchors for coupling the polynucleotide to the membrane, the hairpinloop adaptor comprises one or more second anchors for coupling thepolynucleotide to the membrane and the strength of coupling of thehairpin loop adaptor to the membrane is greater than the strength ofcoupling of the Y adaptor to the membrane.

The invention provides an alternative method in which the anchors areattached to the adaptors after the adaptors have been ligated to thetarget double stranded polynucleotide. The method involves ligating a Yadaptor to one end of the polynucleotide and ligating a hairpin loopadaptor to the other end of the polynucleotide. One or more firstanchors are then attached to the Y adaptor and one or more secondanchors are attached to the hairpin loop adaptor. As above, the strengthof coupling of the hairpin loop adaptor to the membrane is greater thanthe strength of coupling of the Y adaptor to the membrane. The one ormore anchors may be attached to the adaptor is any way, including any ofthose discussed above. The one or more anchors are preferably attachedto the adaptor via hybridisation.

Any methods of ligation may be used. Suitable methods are disclosedabove.

The Y adaptor and hairpin loop adaptor may be any of those discussedabove.

MuA-Based Methods

The invention also provides a method for modifying a target doublestranded polynucleotide for characterisation using a transmembrane porein a membrane, comprising contacting the target polynucleotide with aMuA transposase and a population of double stranded MuA substrates,wherein a proportion of the substrates in the population are Y adaptorscomprising one or more first anchors for coupling the polynucleotide tothe membrane, wherein a proportion of the substrates in the populationare hairpin loop adaptors comprising one or more second anchors forcoupling the polynucleotide to the membrane and wherein the strength ofcoupling of the hairpin loop adaptor to the membrane is greater than thestrength of coupling of the Y adaptor to the membrane, and therebyproducing a plurality of modified double stranded polynucleotides.

The invention provides an alternative method in which the anchors areattached to the substrates after the target double strandedpolynucleotide has been fragmented by the MuA transposase and thesubstrates have been ligated to the fragments. The method involvescontacting the target polynucleotide with a MuA transposase and apopulation of double stranded MuA substrates, wherein a proportion ofthe substrates in the population are Y adaptors and wherein a proportionof the substrates in the population are hairpin loop adaptors. Thetransposase fragments the target polynucleotide and ligates a substrateto one or both ends of the double stranded fragments and therebyproduces a plurality of fragment/substrate constructs. The transposasepreferably fragments the target polynucleotide and ligates a substrateto both ends of the double stranded fragments and thereby produces aplurality of fragment/substrate constructs. The transposase preferablyproduces a plurality of fragment/substrate constructs each comprising aY adaptor at one end and a hairpin loop adaptor at the other end. Themethod also involves attaching to the Y adaptors in the plurality offragment/substrate constructs one or more first anchors and attaching tothe hairpin loop adaptors in the plurality of fragment/substrateconstructs one or more second anchors and thereby producing a pluralityof modified double stranded polynucleotides. The strength of coupling ofeach hairpin loop adaptor to the membrane is greater than the strengthof coupling of each Y adaptor to the membrane.

The Y adaptor and hairpin loop adaptor may be any of those discussedabove.

MuA-based fragmentation a target double stranded polynucleotide isdisclosed in International Application No. PCT/GB2014/052505 (publishedas WO 2015/022544).

The target polynucleotide is contacted with a MuA transposase. Thiscontacting occurs under conditions which allow the transposase tofunction, i.e. to fragment the target polynucleotide and to ligate MuAsubstrates to the one or both ends of the fragments. MuA transposase iscommercially available, for instance from Thermo Scientific (CatalogueNumber F-750C, 20 μL (1.1 μg/μL)). Conditions under which MuAtransposase will function are known in the art.

The target polynucleotide is contacted with a population of doublestranded MuA substrates. The double stranded substrates arepolynucleotide substrates and may be formed from any of the nucleotides,polynucleotides or nucleic acids discussed above.

Each substrate typically comprises a double stranded portion whichprovides its activity as a substrate for MuA transposase. The doublestranded portion is typically the same in each substrate. The populationof substrates may comprise different double stranded portions. Eachsubstrate preferably comprises a double stranded portion which comprisesthe sequence shown in SEQ ID NO: 26 hybridised to a sequence which iscomplementary to the sequence shown in SEQ ID NO: 26. The at least oneoverhang is preferably at the 5′ end of the sequence which iscomplementary to the sequence shown in SEQ ID NO: 26.

In a preferred embodiment, each substrate in the population comprises atleast one overhang of universal nucleotides such that the transposasefragments the target polynucleotide and ligates a substrate to one orboth ends, preferably both ends, of the double stranded fragments andthereby produces a plurality of fragment/substrate constructs andwherein the method further comprises ligating the overhangs to thefragments in the constructs and thereby producing a plurality ofmodified double stranded polynucleotides. The transposase preferablyproduces a plurality of fragment/substrate constructs each comprising aY adaptor at one end and a hairpin loop adaptor at the other end.

Each substrate preferably comprises only one overhang. The only oneoverhang is preferably at the 5′ end of one strand of the doublestranded portion.

The overhang may be at least 3, at least 4, at least 5, at least 6 or atleast 7 nucleotides in length. The overhang is preferably 5 nucleotidesin length.

A universal nucleotide is one which will hybridise to some degree to allof the nucleotides in the target polynucleotide. Suitable universalnucleotides are described in International Application No.PCT/GB2014/052505 (published as WO 2015/022544).

The overhang(s) of universal nucleotides may further comprise a reactivegroup, preferably at the 5′ end. The reactive group may be used toligate the overhangs to the fragments in the constructs as discussedbelow. The reactive group may be used to ligate the fragments to theoverhangs using click chemistry. Suitable reactive groups are disclosedin International Application No. PCT/GB2014/052505 (published as WO2015/022544).

In a further embodiment, the modification method uses a MuA transposaseand a population of MuA substrates each comprising at least one overhangcomprising a reactive group. The overhang(s) may be any length and maycomprise any combination of any nucleotide(s). Suitable lengths andnucleotides are disclosed above. Suitable reactive groups are discussedabove.

In another embodiment, the method comprises contacting the targetpolynucleotide with a population of double stranded MuA substrates eachcomprising (i) at least one overhang and (ii) at least one nucleotide inthe same strand as the at least one overhang which comprises anucleoside that is not present in the target polynucleotide such thatthe transposase fragments the target polynucleotide and ligates asubstrate to one or both ends of the double stranded fragments andthereby produces a plurality of fragment/substrate constructs. A skilledperson can identify nucleosides not present in the target polynucleotideas described in International Application No. PCT/GB2014/052505(published as WO 2015/022544). The transposase preferably fragments thetarget polynucleotide and ligates a substrate to both ends of the doublestranded fragments and thereby produces a plurality offragment/substrate constructs. The transposase preferably produces aplurality of fragment/substrate constructs each comprising a Y adaptorat one end and a hairpin loop adaptor at the other end. The overhangscan be removed from the constructs by selectively removing the at leastone nucleotide to produce a plurality of double stranded constructscomprising single stranded gaps. The single stranded gaps in theconstructs can be repaired to produce a plurality of modified doublestranded polynucleotides.

One strand of the double stranded portion preferably comprises thesequence shown in SEQ ID NO: 26 and the other strand of the doublestranded portion preferably comprises a sequence which is complementaryto the sequence shown in SEQ ID NO: 26 and which is modified to includeat least one nucleotide that is not present in the targetpolynucleotide. This “other strand” further comprises the overhang. In amore preferred embodiment, one strand of the double stranded portioncomprises the sequence shown in SEQ ID NO: 26 and the other strand ofthe double stranded portion comprises the sequence shown in SEQ ID NO:27 (see below). In SEQ ID NO: 27, the dA in the dC and dA dinucleotideat the 3′ end had been replaced with dU. This double stranded portion(shown below) may be used when the target polynucleotide comprisesdeoxyadenosine (dA), thymidine (dT), deoxyguanosine (dG) anddeoxycytidine (dC), but not deoxyuridine (dU).

(SEQ 26) 5′-GTTTTCGCATTTATCGTGAAACGCTTTCGCGTTTTTCGTGCGCCGC TTCA-3′(SEQ 27) 3′-CAAAAGCGTAAATAGCACTTTGCGAAAGCGCAAAAAGCACGCGGCG AAG U -5′

In a most preferred embodiment, one strand of the substrate comprisesthe sequence shown in SEQ ID NO: 26 and the other strand of thesubstrate comprises the sequence shown in SEQ ID NO: 28 (see below).This substrate (shown below) may be used when the target polynucleotidecomprises deoxyadenosine (dA), thymidine (dT), deoxyguanosine (dG) anddeoxycytidine (dC), but not deoxyuridine (dU).

(SEQ 26) 5′-GTTTTCGCATTTATCGTGAAACGCTTTCGCGTTTTTCGTGCGCCGCT TCA-3′(SEQ 28) 3′-CAAAAGCGTAAATAGCACTTTGCGAAAGCGCAAAAAGCACGCGGCGA  AG UCTAG-5′

In all of the embodiments above, the proportion of one type of substratemay be any proportion, such as at least about 5%, at least about 10%, atleast about 20%, at least about 30%, at least about 40%, at least about50%, at least about 60%, at least about 70%, at least about 80%, atleast about 90% or at least about 95%. The remaining proportion ofsubstrates in the population is typically the other type of substrate.For instance, the population may comprise about 40% of the substratescomprising a hairpin loop and about 60% of the Y substrates. Theproportion of both types of substrate is preferably about 50%.

Methods for ligating the overhangs to the fragments, selectivelyremoving the nucleotide(s) which comprise(s) a nucleoside that is notpresent in the target polynucleotide from the ligated constructs andrepairing the single stranded gaps in the double stranded constructs aredisclosed in International Application No. PCT/GB2014/052505 (publishedas WO 2015/022544).

Products of the Invention

The invention also provides a target double stranded polynucleotidemodified using a method of the invention. The modified polynucleotidecomprises a Y adaptor attached at one end of the polynucleotide and ahairpin loop adaptor attached at the other end, wherein the Y adaptorcomprises one or more first anchors for coupling the polynucleotide tothe membrane, wherein the hairpin loop adaptor comprises one or moresecond anchors for coupling the polynucleotide to the membrane andwherein the strength of coupling of the hairpin loop adaptor to themembrane is greater than the strength of coupling of the Y adaptor tothe membrane, and thereby providing a modified target double strandedpolynucleotide.

The invention also provides a plurality of polynucleotides modifiedusing the MuA-based methods of the invention. These modifiedpolynucleotides preferably comprise a Y adaptor attached at one end anda hairpin loop adaptor attached at the other end, wherein the Y adaptorcomprises one or more first anchor for coupling the polynucleotide tothe membrane, wherein the hairpin loop adaptor comprises one or moresecond anchors for coupling the polynucleotide to the membrane andwherein the strength of coupling of the hairpin loop adaptor to themembrane is greater than the strength of coupling of the Y adaptor tothe membrane, and thereby providing a modified target double strandedpolynucleotide. Some of the polynucleotides may have Y adaptors asdefined above at both ends or hairpin loop adaptors as defined above atboth ends.

The invention also provides a pair of adaptors for modifying a targetdouble stranded polynucleotide for characterisation using atransmembrane pore in a membrane, wherein one adaptor is a Y adaptorcomprising one or more first anchors for coupling the polynucleotide tothe membrane, wherein the other adaptor is a hairpin loop adaptorcomprising one or more second anchors for coupling the polynucleotide tothe membrane and wherein the strength of coupling of the hairpin loopadaptors to the membrane is greater than the strength of coupling of theY adaptor to the membrane.

The invention also provides population of adaptors for modifying atarget polynucleotide for characterisation using a transmembrane pore ina membrane, wherein a proportion of the adaptors are Y adaptorscomprising one or more first anchors for coupling the polynucleotide tothe membrane, wherein a proportion of the adaptors are hairpin loopadaptors comprising one or more second anchors for coupling thepolynucleotide to the membrane and wherein the strength of coupling ofthe hairpin loop adaptor to the membrane is greater than the strength ofcoupling of the Y adaptor to the membrane.

Each adaptor in the pair or population of the invention preferablycomprises a double stranded MuA substrate. The substrates may be any ofthose described above. The substrates preferably comprise a doublestranded portion as defined above. The double stranded portionpreferably comprises SEQ ID NOs: 26 and 27 as discussed above. Thedouble stranded portion more preferably comprises SEQ ID NOs: 26 and 28as discussed above.

The proportion values given above in connection with the method of theinvention are equally

applicable to the populations of the invention.

Any of the embodiments discussed above with reference to the methods ofthe invention are equally applicable to the polynucleotides, pairs andpopulations of the invention.

The population or plurality may be isolated, substantially isolated,purified or substantially purified. A population or plurality isisolated or purified if it is completely free of any other components,such as the target polynucleotide, lipids or pores. A population orplurality is substantially isolated if it is mixed with carriers ordiluents which will not interfere with its intended use. For instance, apopulation or plurality is substantially isolated or substantiallypurified if it is present in a form that comprises less than 10%, lessthan 5%, less than 2% or less than 1% of other components, such aslipids or pores.

Kits

The present invention also provides a kit for modifying a targetpolynucleotide comprising (a) a pair of adaptors of the invention or apopulation of adaptors of the invention and (b) a MuA transposase. Eachadaptor in the pair or population preferably comprises a double strandedMuA substrate.

Any of the embodiments discussed above with reference to the methods andproducts of the invention equally apply to the kits.

The kit may further comprise the components of a membrane, such as thecomponents of an amphiphilic layer or a triblock copolymer membrane. Thekit may further comprise a transmembrane pore or the components of atransmembrane pore. The kit may further comprise a polynucleotidebinding protein. Suitable membranes, pores and polynucleotide bindingproteins are discussed above.

The kit of the invention may additionally comprise one or more otherreagents or instruments which enable any of the embodiments mentionedabove to be carried out. Such reagents or instruments include one ormore of the following: suitable buffer(s) (aqueous solutions), means toobtain a sample from a subject (such as a vessel or an instrumentcomprising a needle), means to amplify and/or express polynucleotides, amembrane as defined above or voltage or patch clamp apparatus. Reagentsmay be present in the kit in a dry state such that a fluid sampleresuspends the reagents. The kit may also, optionally, compriseinstructions to enable the kit to be used in the method of the inventionor details regarding which patients the method may be used for. The kitmay, optionally, comprise nucleotides.

The following Example illustrates the invention.

EXAMPLE 1

This example describes the sample preparation procedure for the DNAconstructs 1-6 shown in FIGS. 1 and 2 and used in Examples 2 and 3.

Materials and Methods

The strands used in this study are from a region of the lambda genome,between 45,042 bp and 48,487 bp. Analytes were made by the polymerasePCR method to include hybridisation sites at defined ends of each of thetemplate and template complement strands as desired. PCR was carried outfrom lambda genomic DNA.

This template (SEQ ID NO: 29 hybridised to 30) was made using KAPA HiFi2× Master mix, lambda DNA (NEB) and primers SEQ ID NO: 31 and SEQ ID NO:32. Reactions were cycled 20 times and product of the correct size waspurified by Gel Filtration on Sephacryl S1000 column and concentrated to0.25 mg/ml using Millipore Ultracel 15 50 kDa concentrators.

DNA constructs (1, 2, 3, 4, 5 and 6) for electrophysiology experimentswere all made according to the same reaction mix; 2× LongAmp Taq mastermix, 300 nM of primers 1 and 2 or 3 and 4 (strand labelled a1 in FIGS. 1and 2 was produced using primer 1=SEQ ID NO: 33 attached at its 3′ endto four iSpC3 spacers which are attached at the opposite end to the 5′end of SEQ ID NO: 34 which is attached at its 3′ end to four iSpC3spacers which are attached to four 5-nitroindoles which are attached atthe opposite end to the 5′ end of SEQ ID NO: 36 and primer 2=SEQ ID NO:37 attached at the 3′ end to four 5-nitroindoles which are attached atthe opposite end to the 5′ end of SEQ ID NO: 38, strand labelled a2 inFIGS. 1 and 2 was produced using primer 3=SEQ ID NO: 33 attached at its3′ end to four iSpC3 spacers which are attached at the opposite end tothe 5′ end of SEQ ID NO: 37 which is attached at its 3′ end to fouriSpC3 spacers which are attached to four 5-nitroindoles which areattached at the opposite end to the 5′ end of SEQ ID NO: 38 and primer4=SEQ ID NO: 34 attached at the 3′ end to four 5-nitroindoles which areattached at the opposite end to the 5′ end of SEQ ID NO: 36) and 1.2 ngul⁻¹ DNA template (SEQ ID NO: 29 hybridised to 30). DNA constructs wereall amplified according to the same cycling program; 94° C. for 2 mins,[94° C. for 15 secs, 58° C. for 30 secs, 65° C. for 2 mins]₁₂ and 65° C.for 5 mins. DNA constructs were all purified from a 0.8% agarose gelaccording to manufacturer's instructions (Qiagen Gel Extraction kit) andthen SPRI purified (Agencourt AMPure beads) according to manufacturer'sinstructions.

Finally, the DNA strands produced were hybridised to complementarystrands of DNA, some of which contained anchors e.g. cholesterol orpalmitate. The complementary strands SEQ ID NO: 39 and 40 (with andwithout attached anchors) were annealed at a five-fold excess at roomtemperature for ten minutes in 25 mM potassium phosphate buffer, 151 mMpotassium chloride, pH 8.0.

DNA construct 1 was made up of four different strands hybridisedtogether as shown in FIG. 1(A)—a1=SEQ ID NO: 33 attached at its 3′ endto four iSpC3 spacers which are attached at the opposite end to the 5′end of SEQ ID NO: 34; SEQ ID NO: 34 is attached at the 3′ end to fouriSpC3 spacers which are attached at the opposite end to four5-nitroindoles which are attached to the 5′ end of SEQ ID NO: 35, a3=SEQID NO: 40 which is attached at its 3′ end to six iSp18 spacers which areattached at the opposite end to two thymines which are attached at theopposite end to a 3′ cholesterol TEG, a4=SEQ ID NO: 39 anda5=complementary sequence to a3 and part of a1.

DNA construct 2 was made up of four different strands hybridisedtogether as shown in FIG. 1(A) and (B)—a2=SEQ ID NO: 33 attached at its3′ end to four iSpC3 spacers which are attached at the opposite end tothe 5′ end of SEQ ID NO: 37; SEQ ID NO: 37 is attached at the 3′ end tofour iSpC3 spacers which are attached at the opposite end to four5-nitroindoles which are attached to the 5′ end of SEQ ID NO: 41, a6=SEQID NO: 39 which is attached at its 3′ end to six iSp18 spacers which areattached at the opposite end to two thymines which are attached at theopposite end to a 3′ cholesterol TEG, a7=SEQ ID NO: 40 anda8=complementary sequence to a6 and part of a2.

DNA construct 3 was made up of four different strands hybridisedtogether as shown in FIG. 1(B) and 2(B)—a2=SEQ ID NO: 33 attached at its3′ end to four iSpC3 spacers which are attached at the opposite end tothe 5′ end of SEQ ID NO: 34; SEQ ID NO: 34 is attached at the 3′ end tofour iSpC3 spacers which are attached at the opposite end to four5-nitroindoles which are attached to the 5′ end of SEQ ID NO: 35, a3=SEQID NO: 40 which is attached at its 3′ end to six iSp18 spacers which areattached at the opposite end to two thymines which are attached at theopposite end to a 3′ cholesterol TEG, a9=SEQ ID NO: 39 which is attachedat its 3′ end to six iSp18 spacers which are attached at the oppositeend to two thymines which are attached at the opposite end to a 3′palmitate and a5=complementary sequence to a3 and part of a1.

DNA construct 4 was made up of four different strands hybridisedtogether as shown in FIG. 2(A)—a1=SEQ ID NO: 33 attached at its 3′ endto four iSpC3 spacers which are attached at the opposite end to the 5′end of SEQ ID NO: 34; SEQ ID NO: 34 is attached at the 3′ end to fouriSpC3 spacers which are attached at the opposite end to four5-nitroindoles which are attached to the 5′ end of SEQ ID NO: 35, a3=SEQID NO: 40 which is attached at its 3′ end to six iSp18 spacers which areattached at the opposite end to two thymines which are attached at theopposite end to a 3′ cholesterol TEG, a6=SEQ ID NO: 39 which is attachedat its 3′ end to six iSp18 spacers which are attached at the oppositeend to two thymines which are attached at the opposite end to a 3′cholesterol TEG and a5=complementary sequence to a3 and part of a1.

DNA construct 5 was made up of four different strands hybridisedtogether as shown in FIG. 2(A)—a1=SEQ ID NO: 33 attached at its 3′ endto four iSpC3 spacers which are attached at the opposite end to the 5′end of SEQ ID NO: 34; SEQ ID NO: 34 is attached at the 3′ end to fouriSpC3 spacers which are attached at the opposite end to four5-nitroindoles which are attached to the 5′ end of SEQ ID NO: 35, a3=SEQID NO: 40 which is attached at its 3′ end to six iSp18 spacers which areattached at the opposite end to two thymines which are attached at theopposite end to a 3′ cholesterol TEG, a2=SEQ ID NO: 33 attached at its3′ end to four iSpC3 spacers which are attached at the opposite end tothe 5′ end of SEQ ID NO: 37; SEQ ID NO: 37 is attached at the 3′ end tofour iSpC3 spacers which are attached at the opposite end to four5-nitroindoles which are attached to the 5′ end of SEQ ID NO: 41 anda6=SEQ ID NO: 39 which is attached at its 3′ end to six iSp18 spacerswhich are attached at the opposite end to two thymines which areattached at the opposite end to a 3′ cholesterol TEG.

DNA construct 6 was made up of four different strands hybridisedtogether as shown in FIG. 2(B)—a1=SEQ ID NO: 33 attached at its 3′ endto four iSpC3 spacers which are attached at the opposite end to the 5′end of SEQ ID NO: 34; SEQ ID NO: 34 is attached at the 3′ end to fouriSpC3 spacers which are attached at the opposite end to four5-nitroindoles which are attached to the 5′ end of SEQ ID NO: 35,a10=SEQ ID NO: 40 which is attached at its 3′ end to six iSp18 spacerswhich are attached at the opposite end to two thymines which areattached at the opposite end to a 3′ palmitate, a2=SEQ ID NO: 33attached at its 3′ end to four iSpC3 spacers which are attached at theopposite end to the 5′ end of SEQ ID NO: 37; SEQ ID NO: 37 is attachedat the 3′ end to four iSpC3 spacers which are attached at the oppositeend to four 5-nitroindoles which are attached to the 5′ end of SEQ IDNO: 41 and a9=SEQ ID NO: 39 which is attached at its 3′ end to six iSp18spacers which are attached at the opposite end to two thymines which areattached at the opposite end to a 3′ cholesterol TEG.

EXAMPLE 2

This example compares the use of a single anchor to couple constructs toa membrane to that of two anchors of differing strengths again used tocouple constructs to a membrane. Two anchors were employed in order tobias, the helicase-controlled DNA movements detected by the nanoporesystem, towards the construct which was doubly coupled to the membrane.

Materials and Methods

Prior to setting up the experiment, the DNA constructs either 1 and 2 or2 and 3 (stock concentration 20 nM, final concentration added tonanopore system 0.1 nM) were separately pre-incubated at roomtemperature for five minutes with T4 Dda-E94C/A360C (stock concentration250 nM, final concentration added to nanopore system 1 nM , SEQ ID NO:24 with mutations E94C/A360C) in buffer (151 mM KCl, 25 mM phosphate, 2mM EDTA, pH8.0). After five minutes, TMAD (500 μM) was added to thepre-mix and the mixture incubated for a further 5 minutes. Finally,MgCl2 (10 mM final concentration), ATP (2.5 mM final concentration) andbuffer (150 mM potassium ferrocyanide (II), 150 mM potassiumferricyanide and 25 mM potassium phosphate pH 8.0) were added to thepre-mix.

Electrical measurements were acquired from single MspA nanopores(MspA-B2C) inserted in block co-polymer in buffer (25 mM potassiumphosphate, 150 mM potassium ferrocyanide (II), 150 mM potassiumferricyanide (III), pH 8.0). After achieving a single pore inserted inthe block co-polymer, then buffer (2 mL, 25 mM potassium phosphate pH8.0, 150 mM potassium ferrocyanide (II) and 150 mM potassiumferricyanide (III)) was flowed through the system to remove any excessMspA nanopores. The enzyme (T4 Dda-E94C/A360C, 1 nM finalconcentration), DNA construct either 1 and 2 or 2 and 3 (0.1 nM finalconcentration), fuel (MgCl2 10 mM final concentration, ATP 2.5 mM finalconcentration) pre-mix (150 μL total) was then added to the singlenanopore experimental system and the experiment run at a holdingpotential of 120 mV for 2 hours and helicase-controlled DNA movementmonitored.

Results and Discussion

For the control experiment, helicase-controlled DNA movement ofconstructs 1 and 2 was monitored. The cartoon at the top of FIG. 3 showswhere the helicase can bind to constructs 1 and 2. Helicase controlledDNA movements corresponding to each construct were identified and theproportion of movements corresponding to strand a1 (construct 1) andstrand a2 (construct 2) were compared (see the bottom half of FIG. 3).As each construct was coupled to the membrane using the same anchor (acholesterol anchor) then, of the helicase-controlled DNA movementsobserved, approximately 50% corresponded to strand a1 and 50% to stranda2. This illustrated that by using the same single anchor, there was nobias towards helicase-controlled translocation movements for oneconstruct over the other.

The experiment also compared the use of a single anchor (cholesterol) tocouple construct 2 to the membrane, to the use of two different anchors(of differing strength—cholesterol and palmitate) to couple construct 3to the membrane. The cartoon at the top of FIG. 4 shows where thehelicase can bind to strand a1 (constructs 3) and strand a2 (construct2). Helicase controlled DNA movements corresponding to each constructwere identified and the proportion of movements corresponding to a1 anda2 (construct 3 and 2 respectively) were compared (see the bottom halfof FIG. 4). In this case more than 95% of the helicase-controlled DNAmovements detected corresponded to strand a1 (construct 3), which wascoupled to the membrane using a cholesterol and a palmitate. Less than5% of the helicase-controlled DNA movements corresponded to strand a2(construct 2). This experiment illustrated that by using two differentanchors it was possible to strongly bias the helicase-controlled DNAmovements detected by the nanopore system towards the doubly coupledconstruct over the singly coupled construct.

EXAMPLE 3

This example compares the use of two anchors of equal strength (doublecholesterol constructs 4 and 5 or double palmitate construct 6) to twoanchors of differing strengths (palmitate and cholesterol construct 3).

Materials and Methods

DNA constructs 4 and 5 or 3 and 6 were pre-incubated with T4Dda-E94C/A360C helicase as described in Example 2 above.

Electrical measurements were acquired from single MspA nanopores(MspA-B2C) inserted in block co-polymer in buffer as described inExample 2 and helicase-controlled DNA movements for constructs 4 and 5or 3 and 6 were monitored.

Results and Discussion

For the control experiment, helicase-controlled DNA movement ofconstructs 4 and 5 was monitored. The cartoon at the top of FIG. 5 showswhere the helicase can bind to constructs 4 (only on a1) and 5 (on a1and a2). Strand a1 of construct 4 was capable of being captured by thenanopore, whereas, strand a1 or strand a2 of construct 5 were capable ofbeing captured by the nanopore. Helicase controlled DNA movementscorresponding to each construct were identified and the proportion ofmovements corresponding to strand a1 (construct 4 and 5) and strand a2(construct 5 only) were compared (see the bottom half of FIG. 5). Aseach construct was coupled to the membrane using the same anchor (twocholesterol anchors) then of the helicase-controlled DNA movementsobserved, approximately 66% corresponded to strand a1 (construct 4 and5) and 33% to strand a2 (construct 5 only). The 2:1 bias towards thedetection of a1 helicase controlled DNA movements was expected becausea2 movements would only be detected if construct 4 was captured by thenanopore, whereas a1 movements would be detected from capture of bothconstruct 4 and 5, therefore, twice as many a1 movements were expected(as equivalent concentrations of constructs 4 and 5 were added to thenanopore experimental system).

The experiment also compared the use of two coupling agents of differingstrengths (cholesterol and palmitate) to couple construct 3 to themembrane, to the use of two identical coupling agents (both palmitate)to couple construct 6 to the membrane. The cartoon at the top of FIG. 6shows where the helicase can bind to strand a1 (constructs 3 and 6) andstrand a2 (construct 6 only). Strand a1 of construct 3 was capable ofbeing captured by the nanopore, whereas, strand a1 or strand a2 ofconstruct 6 were capable of being captured by the nanopore. Helicasecontrolled DNA movements corresponding to each construct were identifiedand the proportion of movements corresponding to a1 (construct 2 and 3)and a2 (construct 2 only) were compared (see the bottom half of FIG. 6).In this case more than 95% of the helicase-controlled DNA movementsdetected corresponded to strand a2 (construct 3 and 6), which werecoupled to the membrane using either a cholesterol and a palmitate ortwo palmitates. Less than 5% of the helicase-controlled DNA movementscorresponded to strand a2 (construct 6 only). This experimentillustrated that by using two different coupling agents it was possibleto strongly bias the helicase-controlled DNA movements detected by thenanopore system towards the construct coupled by coupling agents ofdiffering strength rather than the construct coupled by two identicalcoupling agents of equal strength. This also shows that by using thecombination of a stronger and weaker anchor on a construct that can onlybe captured by the nanopore at one end it was possible to bias towardsthis construct over a construct that has two weaker anchors on aconstruct that can be captured by the nanopore at either end.

EXAMPLE 4

This example shows how the relative strengths of a number of couplingagents can be compared. This would allow selection of appropriatestrength coupling agents for coupling of a construct to a membrane.

Materials and Methods

DNA constructs 1, 2 and 7(a-c) were produced using the method describedin Example 1. Constructs 1 and 2 are described in detail above andconstructs 7(a-c) are described below.

DNA construct 7a was made up of four different strands hybridisedtogether as shown in FIG. 7(B)—a2=SEQ ID NO: 33 attached at its 3′ endto four iSpC3 spacers which are attached at the opposite end to the 5′end of SEQ ID NO: 37; SEQ ID NO: 37 is attached at the 3′ end to fouriSpC3 spacers which are attached at the opposite end to four5-nitroindoles which are attached to the 5′ end of SEQ ID NO: 41,a10(a)=SEQ ID NO: 42 which has six iSp18 spacers attached to its 5′ endwhich are attached at the opposite end to two thymines and a 5′cholesterol TEG; SEQ ID NO: 42 has a further six iSp18 spacers attachedto its 3′ end which are attached at the opposite end to two thymines anda 3′ cholesterol TEG, a7=SEQ ID NO: 40 and a8=complementary sequence toA10a and part of a2 .

DNA construct 7b was made up of four different strands hybridisedtogether as shown in FIG. 7(B)—a2=SEQ ID NO: 33 attached at its 3′ endto four iSpC3 spacers which are attached at the opposite end to the 5′end of SEQ ID NO: 37; SEQ ID NO: 37 is attached at the 3′ end to fouriSpC3 spacers which are attached at the opposite end to four5-nitroindoles which are attached to the 5′ end of SEQ ID NO: 41,a10(b)=SEQ ID NO: 39 which is attached at its 3′ end to six iSp18spacers which are attached at the opposite end to two thymines which areattached at the opposite end to a 3′ tocopherol, a7=SEQ ID NO: 40 anda8=complementary sequence to A10b and part of a2 .

DNA construct 7c was made up of four different strands hybridisedtogether as shown in FIG. 7(B)—a2=SEQ ID NO: 33 attached at its 3′ endto four iSpC3 spacers which are attached at the opposite end to the 5′end of SEQ ID NO: 37; SEQ ID NO: 37 is attached at the 3′ end to fouriSpC3 spacers which are attached at the opposite end to four5-nitroindoles which are attached to the 5′ end of SEQ ID NO: 41,a10(c)=SEQ ID NO: 39 which is attached at its 3′ end to six iSp18spacers which are attached at the opposite end to two thymines which areattached at the opposite end to a 3′ palmitate, a7=SEQ ID NO: 40 anda8=complementary sequence to A10c and part of a2 .

DNA constructs 1, 2 and 7(a-c) were pre-incubated with T4 Dda-E94C/A360C helicase as described in Example 2 above.

Electrical measurements were acquired from single MspA nanopores (MspA−B2C) inserted in block co-polymer in buffer as described in Example 2and helicase-controlled DNA movements for constructs 1, 2 and 7(a-c)were monitored.

Results and Discussion

For the control experiment, helicase-controlled DNA movement ofconstructs 1 and 2 was monitored. The cartoon at the top of FIG. 8 showswhere the helicase can bind to constructs 1 and 2. Helicase controlledDNA movements corresponding to each construct were identified and theproportion of movements corresponding to strand a1 (construct 1) andstrand a2 (construct 2) were compared (see the bottom half of FIG. 8).As each construct was coupled to the membrane using the same couplingagent cholesterol) then, of the helicase-controlled DNA movementsobserved, approximately 50% corresponded to strand a1 and 50% to stranda2 This illustrated that by using the same single coupling agent, therewas no bias towards helicase-controlled translocation movements for oneconstruct over the other.

The experiment also compared the use of a variety of different couplingagents to couple constructs 7(a-c) to the membrane to that ofcholesterol which was used to couple construct 1 to the membrane. Thecartoons at the top of FIGS. 9-11 show where the helicase could bind tostrand a1 (constructs 1) and strand a2 (construct 7(a-c)). Helicasecontrolled DNA movements corresponding to each construct were identifiedand the proportion of movements corresponding to a1 and a2 (construct 1and 7(a-c) respectively) were compared (see the bottom half of FIGS.9-11). The use of two cholesterols in the same strand of DNA (A10a) wasobserved to have an equivalent coupling strength to that of the singlecholesterol (see FIG. 9). Whereas, the use of a tocopherol was observedto be a stronger coupling agent than cholesterol (see FIG. 10), as agreater number of DNA controlled helicase movements were observed forthe construct anchored by tocopherol than cholesterol. Furthermore,palmitate was observed to be a weaker coupling agent than cholesterolbecause fewer helicase controlled DNA movements were observed for theconstruct anchored by palmitate than cholesterol (see FIG. 11).Therefore, by comparing the coupling strengths of various couplingagents using this method it would be possible to select two differentstrength coupling agents and bias the system towards selection of adesired construct (see example 6 for further details).

EXAMPLE 5

This example describes the sample preparation procedure for the DNAconstructs 8 and 9 shown in FIG. 12 and used in Examples 6.

Materials and Methods

Lambda DNA (1 μg, SEQ ID NO: 43) was restriction enzyme digested for 1hour at 37° C. in 1× NEBuffer 4, with 5 U of SnaBI and 20 U of BamHI-HF.Following digestion, the DNA was purified using 1.5× Agencourt AMPurebeads, according to the manufacturer's protocol. The sample was theneluted in 50 uL of nuclease free water. The DNA was then end-repairedusing NEB's NEBNext End-repair system, following the manufacturer'sprotocol. The sample was again purified using 1.5× Agencourt AMPurebeads and the DNA eluted in 42 uL of nuclease free water. Following theend-repair step, the purified DNA was dA-tailed using NEB's NEBNextdA-tailing system, according to the manufacturer's protocol. The DNA wasagain purified using 1.5× Agencourt AMPure beads, eluting in 20 uL ofnuclease free water. The DNA was quantified at A260 nm and adaptersligated at a 25-fold excess per 5′ end, using NEB's NEBNext quick ligasemodule following the manufacturer's protocol. Construct 8 was producedby ligating the following adapters to the fragments of DNA (labelled asa12 in FIG. 12) (adapter 1 (labelled a11 in FIG. 12)=25 iSpC3 spacersattached at to the 5′ to SEQ ID NO: 44 which is attached at its 3′ endto four iSp18 spacers which are attached at the opposite end to the 5′end of SEQ ID NO: 45 which is attached at the 3′ end to the sequenceAACCT (which are joined together by phosphorothioate bonds) where thefinal T in the sequence is attached to a further non-modified T; adapter2 (labelled as a14 in FIG. 12)=has a phosphate group at the 5′ end ofsequence GGTT (which are joined together by phosphorothioate bonds),this sequence is attached at its 3′ end to the 5′ end of SEQ ID NO: 46which is attached at the 3′ end to six iSp18 spacers which are attachedat the opposite end to two thymines which are attached at the oppositeend to a 3′ cholesterol TEG and adapter 3 (labelled as a13 in FIG.12)=has a phosphate group at the 5′ end of SEQ ID NO: 47 and which hasan internal cholesterol TEG attached to the 3′ end of SEQ ID NO: 47, theinternal cholesterol is also attached to the 5′ end of SEQ ID NO: 48which is attached at its 3′ end to a G with a phosphorothioate bond anda non-modified T). Construct 9 was produced by ligating the followingadapters to the fragments of DNA (labelled as a12 in FIG. 12) (adapter 1(labelled as a11 in FIG. 12)=25 iSpC3 spacers attached at to the 5′ toSEQ ID NO: 44 which is attached at its 3′ end to four iSp18 spacerswhich are attached at the opposite end to the 5′ end of SEQ ID NO: 45which is attached at the 3′ end to the sequence AACCT (which are joinedtogether by phosphorothioate bonds) where the final T in the sequence isattached to a further non-modified T; adapter 4 (labelled as a15 in FIG.12)=has a phosphate group at the 5′ end of sequence GGTT (which arejoined together by phosphorothioate bonds), this sequence is attached atits 3′ end to the 5′ end of SEQ ID NO: 46 which is attached at the 3′end to six iSp18 spacers which are attached at the opposite end to twothymines which are attached at the opposite end to a 3′ palmitate andadapter 3 (labelled as a13 in FIG. 12)=has a phosphate group at the 5′end of SEQ ID NO: 47 and which has an internal cholesterol TEG attachedto the 3′ end of SEQ ID NO: 47, the internal cholesterol is alsoattached to the 5′ end of SEQ ID NO: 48 which is attached at its 3′ endto a G with a phosphorothioate bond and a non-modified T). Followingdigestion, the ligated DNA was purified using 0.4× Agencourt AMPurebeads, eluting in 16 uL of nuclease free water. The DNA was quantifiedat A260 nm and 4 uL of 757.5 mM KCl, 125 mM potassium phosphate buffer(pH 7) and 5 mM EDTA was added.

EXAMPLE 6

This example compares the helicase controlled DNA movements detected forconstructs 8 and 9. Construct 8 was coupled to the membrane using twocholesterols (both strong coupling agents). Construct 9 was coupled tothe membrane using cholesterol in the hairpin and a palmitate in theY-adaptor. The constructs both contained lambda DNA which had beenfragmented by the process described above.

Materials and Methods

DNA constructs 8 and 9 were each separately pre-incubated with T4Dda—E94C/A360C/C109A/C136A helicase using a similar procedure to thatdescribed in Example 2 above.

Electrical measurements were acquired from single MspA nanopores(MspA-B2C) inserted in block co-polymer in buffer as described inExample 2 and helicase-controlled DNA movements for constructs 8 and 9were monitored.

Results and Discussion

During the fragmentation and adapter attachment process (described inExample 5) the desired constructs shown in FIG. 12 were produced.However, other constructs were also produced during that process whichhad either two Y-adapters attached (constructs 11 and 12) or two hairpinadapters attached (construct 10) (see FIGS. 13 and 14). In order for thenanopore to be able to capture DNA, the DNA must have had at least oneY-adapter so that there was a free end to be captured by the pore.Therefore, the DNA constructs with two hairpins attached (construct 10)were not be observed in the nanopore experiments. The constructs whichhad a Y-adapter on both ends of the lambda DNA fragments could becaptured by the nanopore by either end of the DNA (constructs 11 and12). However, as there was no hairpin attached to the DNA, then onlystrand x or strand y was translocated through the nanopore. However, ifconstructs 8 or 9 were captured by the nanopore (via the Y-adapter end)then both strand X and strand Y translocated through the nanopore owingto the hairpin attached at the opposite end. Therefore, it was desiredthat the constructs which contained a Y adapter and a hairpin werepreferentially captured by the nanopore. It was not possible to separateout the various constructs (10, 8 and 11 or 10, 9 and 12) in order toisolate just the desired constructs 8 and 9.

It was investigated as to whether constructs which contained a Y-adapterand a hairpin adapter could be preferentially selected and captured bythe nanopore by utilising different combinations of coupling agents. Inthis example coupling of two cholesterols (construct 8) was compared tocoupling of cholesterol and a palmitate (construct 9). Both of theconstructs tested exhibited high numbers of helicase controlled DNAmovements of either the hairpin/Y-adapter constructs (8 and 9) or thedouble Y-adapter constructs (11 and 12). When the same coupling agent(cholesterol) was used in both the Y-adapter and the hairpin (construct8) then 25% of the helicase-controlled DNA movements detectedcorresponded to construct 8 and 75% to construct 11. Therefore, only aquarter of the movements detected corresponded to the desired construct.When a strong coupling agent (cholesterol in this case) was used in thehairpin and a weaker coupling agent was used in the Y-adapter (palmitatein this case) 46% of the helicase-controlled DNA movements detectedcorresponded to construct 9 and 56% to construct 12. This is asignificant improvement towards selection of the desired construct 9.This improvement in detection of the desired construct was owing to theuse of two coupling agents of differing strengths. The double Y-adapterconstruct 12 was coupled to the membrane using two weaker couplingagents in comparison to the Y-adapter/hairpin construct 9 which wascoupled to the membrane using one weak and one strong anchor. As thedesired construct employed one anchor which was stronger than those usedin the double Y-adapter construct this was preferentially coupled to themembrane and therefore a larger proportion of the helicase-controlledDNA movements detected corresponded to desired construct 9.

EXAMPLE 7

This example compares the coupling efficiency of a number of differentDNA constructs 13, 14, 15 and 16 which were coupled to the membrane by anumber of means. Construct 13 was coupled to the membrane usingcholesterol in the hairpin region and a palmitate hybridised by a rigidDNA linker in the Y-adaptor region. Construct 14 was coupled to themembrane using cholesterol in the hairpin region and a palmitatehybridised by a flexible DNA linker in the Y-adaptor region. Construct15 was coupled to the membrane using cholesterol in the hairpin regionand a cholesterol hybridised by a rigid DNA linker in the Y-adaptorregion. Construct 16 was coupled to the membrane using cholesterol inthe hairpin region and a cholesterol hybridised by a flexible DNA linkerin the Y-adaptor region.

Materials and Methods

The constructs all contained lambda DNA fragments (labelled in FIGS. 15and 16 as a12) which had been fragmented by the process described abovein Example 5, however, different adapters were then ligated to the DNAconstructs. The adapters ligated to form DNA constructs 13-16 (shown inFIGS. 15 and 16) are described in detail below—

Constructs 13, 14, 15 and 16 were produced by ligating the followingadapters to the fragments of lambda DNA (labelled as a12 in FIGS. 15 and16) (adapter 1 (labelled a16 in FIG. 15)=30 iSpC3 spacers attached tothe 5′ to SEQ ID NO: 51 which is attached at its 3′ end to four iSp18spacers which are attached at the opposite end to the 5′ end of SEQ IDNO: 52; adapter 2 (labelled as a17 in FIG. 15)=has a phosphate group atthe 5′ end of SEQ ID NO: 49, this sequence is attached at its 3′ end tofour iSpC3 spacer which are attached at the opposite end to the 5′ endof SEQ ID NO: 50 which is attached at the 3′ end to a Thymine via aphosphothioate bond; and adapter 3 (labelled as a18 in FIG. 15)=has aphosphate group at the 5′ end of SEQ ID NO: 53.

After purification of the above constructs as described in Example 5then either rigid or flexible DNA linkers with either palmitate orcholesterol attached at the 5′ end were then hybridised to theconstructs under the following conditions—0.2ul of each DNA linker (10uM) was added to 13 ul of ligated DNA (stock conc. approximately 20-30nM) and incubated at room temperature for 10 mins.

Construct 13 was hybridised to the adaptor 4 (labelled in FIG. 15 asa20)=SEQ ID NO: 40 is attached at its 5′ end to six iSp18 spacers whichare attached at the opposite end to two thymines and a 5′ cholesterolTEG and adaptor 5 (labelled in FIG. 15 as a19)=SEQ ID NO: 54 which isattached at the 5′ end to a palmitate.

Construct 14 was hybridised to the adaptor 4 (labelled in FIG. 15 asa20)=SEQ ID NO: 40 is attached at its 5′ end to six iSp18 spacers whichare attached at the opposite end to two thymines and a 5′ cholesterolTEG and adaptor 6 (labelled in FIG. 15 as a21)=SEQ ID NO: 54 which isattached at its 5′ end to six iSp18 spacers which are attached at theopposite end to two thymines and a palmitate.

Construct 15 was hybridised to the adaptor 4 (labelled in FIG. 15 asa20)=SEQ ID NO: 40 is attached at its 5′ end to six iSp18 spacers whichare attached at the opposite end to two thymines and a 5′ cholesterolTEG and adaptor 5 (labelled in FIG. 15 as a22)=SEQ ID NO: 54 which isattached at the 5′ end to a cholesterol TEG.

Construct 16 was hybridised to the adaptor 4 (labelled in FIG. 15 asa20)=SEQ ID NO: 40 is attached at its 5′ end to six iSp18 spacers whichare attached at the opposite end to two thymines and a 5′ cholesterolTEG and adaptor 6 (labelled in FIG. 15 as a23)=SEQ ID NO: 54 which isattached at its 5′ end to six iSp18 spacers which are attached at theopposite end to two thymines and a cholesterol TEG.

Prior to setting up the experiment, the DNA constructs 13-16 (finalconcentration added to nanopore system 0.2 nM) were separatelypre-incubated at room temperature for five minutes with T4Dda-E94C/A360C/C109A/C136A (final concentration added to nanopore system10 nM) in buffer (151 mM KCl, 25 mM phosphate, 5% glycerol, pH7.0).After five minutes, TMAD (500 μM) was added to the pre-mix and themixture incubated for a further 5 minutes. Finally, MgCl2 (2 mM finalconcentration), ATP (2.5 mM final concentration) and buffer (500 mM KCland 25 mM potassium phosphate pH 8.0) were added to the pre-mix.

Electrical measurements were acquired from MspA nanopores (MspA-B2C)inserted in block co-polymer in buffer as described in Example 2 andhelicase-controlled DNA movements for constructs 13-16 were monitored.

Results and Discussion

This example compares the coupling efficiency of a number of differentDNA constructs 13, 14, 15 and 16 by investigating the number of helicasecontrolled DNA movements which were observed for each nanopore over thecourse of an experiment and the percentage of these helicase-controlledmovements which corresponded to translocation of both region R1 and R2when compared to all other helicase-controlled DNA movements detected.

For all four of the different constructs tested, helicase controlled DNAmovements were observed. In this experiment we have compared thestrengths of two different types of anchor—palmitate and cholesterol. Itwas also possible to compare how the flexibility of two different typesof double-stranded polynucleotide linkers affected the strength of thecoupling. In all four constructs, the coupling agent in the hairpin a20(flexible cholesterol tether) remained constant and the coupling agentused in the Y-adapter was varied between palmitate/cholesterol andflexible/rigid linkers. The constructs used in this experiment were madeby the same fragmentation and adapter attachment process described inExample 5. This produced the desired constructs shown in FIGS. 15 and 16as well as constructs analogous to those shown in FIGS. 13 and 14 whichhad either two Y-adapters attached or two hairpin adapters attached. Asdescribed in more detail in example 6 above, in order for the nanoporeto be able to capture DNA, the DNA must have had at least one Y-adapterso that there was a free end which was able to be captured by the pore.Therefore, DNA constructs with two hairpins attached were not observedin the nanopore experiments. The constructs which had a Y-adapter onboth ends of the DNA fragments were captured by the nanopore by eitherend of the DNA. However, as there was no hairpin attached to the DNA,then only R1 or R2 would have been translocated through the nanopore.However, if the desired constructs β-16 were captured by the nanopore(via the Y-adapter end) then regions R1 and R2 would have bothtranslocated through the nanopore. Therefore, it was desired that theconstructs which contained a Y adapter and a hairpin were preferentiallycaptured by the nanopore.

The data shown in FIG. 17 illustrates the % of the total number ofhelicase controlled DNA movements detected which corresponded to thetranslocation of both R1 and R2 regions through the nanopore (which areproduced from the desired constructs which contained a Y-adapter and ahairpin adapter). Other helicase-controlled DNA movements which wereobserved corresponded to translocation of constructs which had twoY-adapters attached to the fragments of lambda DNA rather than aY-adapter and a hairpin adapter. The highest percentage ofhelicase-controlled DNA movements of both R1 and R2 regions through thenanopore (and corresponded to the desired construct) was observed when apalmitate was used with either a rigid or flexible linker (constructs 13and 14). The cholesterol with the rigid tether had a higher percentageof helicase-controlled DNA movements which corresponded to translocationof both R1 and R2 through the nanopore than the cholesterol with theflexible tether.

It is also clear from the data shown in FIG. 18 that for both palmitate(constructs 13 and 14) and cholesterol (constructs 15 and 16) the rigidarrangement was a slightly weaker coupling arrangement than the flexiblearrangement as constructs 13 and 15 resulted in fewerhelicase-controlled DNA movements detected per nanopore. The weakestcoupling arrangement tested was in construct 15 where a rigid linkerattached to a palmitate was used.

The preferred coupling system would be one which resulted in a highnumber of helicase-controlled DNA movements per nanopore which also hadthe highest percentage of helicase-controlled DNA movements whichcorresponded to translocation of regions R1 and R2. The experimentsshowed that the highest % of helicase controlled DNA movements for R1and R2 resulted in the lowest number of helicase-controlled DNAmovements per nanopore and vice versa. Therefore, the preferredconstruct design for optimum overall throughput of the desired helicasecontrolled DNA movements was construct 15 which had a high number ofhelicase-controlled DNA movements per nanopore with a percentage ofhelicase-controlled DNA movements which corresponded to translocation ofregions R1 and R2 of just under 50%.

1. A method of characterising a target double stranded polynucleotideusing a transmembrane pore in a membrane, comprising: a) providing thetarget double stranded polynucleotide with a Y adaptor at one end and ahairpin loop adaptor at the other end, wherein the Y adaptor comprisesone or more first anchors for coupling the polynucleotide to themembrane, wherein the hairpin loop adaptor comprises one or more secondanchors for coupling the polynucleotide to the membrane and wherein thestrength of coupling of the hairpin loop adaptor to the membrane isgreater than the strength of coupling of the Y adaptor to the membrane;b) contacting the polynucleotide provided in step a) with thetransmembrane pore such that at least one strand of the polynucleotidemoves through the pore; and c) taking one or more measurements as the atleast one strand of the polynucleotide moves with respect to the porewherein the measurements are indicative of one or more characteristicsof the at least one strand of the polynucleotide and therebycharacterising the double stranded target polynucleotide.
 2. A methodaccording to claim 1, wherein (a) the hairpin loop adaptor comprisesmore anchors than the Y adaptor, (b) the strength of coupling of one ormore second anchors to the membrane is greater than the strength ofcoupling of the one or more first anchors to the membrane (c) thestrength of coupling of the one or more second anchors to the hairpinloop adaptor is greater than the strength of coupling of the one or morefirst anchors to the Y adaptor, (d) the one or more first anchors andone or more second anchors couple their respective adaptors to themembrane via hybridisation and the strength of hybridisation is greaterin the one or more second anchors than in the one or more first anchors,(e) the one or more first anchors comprise one or more rigid linkers andthe one or more second anchors comprise one or more flexible linkers or(f) any combination of (a) to (e).
 3. A method according to claim 2,wherein (i) the one or more second anchors couple to the membrane usingcholesterol and the one or more first anchors couple to the membraneusing palmitate, (ii) the one or more second anchors couple to themembrane using a mono-acyl species and the one or more first anchorscouple to the membrane using a diacyl species, (iii) the one or morefirst anchors comprise one or more polynucleotide linkers and the one ormore second anchors comprise one or more flexible linkers comprising oneor more one or more spacer 9 (iSp9) groups or one or more spacer 18(iSp18), (iv) a combination of (i) and (iii) or (v) a combination of(ii) and (iii).
 4. A method according to claim 1, wherein step a)comprises modifying the target double stranded polynucleotide so that itcomprises the Y adaptor at one end and the hairpin loop adaptor at theother end.
 5. A method according to claim 1, wherein step b) comprisescontacting the polynucleotide provided in step a) with a transmembranepore such that both strands of the polynucleotide move through the poreand step c) comprises taking one or more measurements as the bothstrands of the polynucleotide move with respect to the pore wherein themeasurements are indicative of one or more characteristics of thestrands of the polynucleotide and thereby characterising the targetpolynucleotide.
 6. A method according to claim 1, wherein the one ormore characteristics are selected from (i) the length of thepolynucleotide, (ii) the identity of the polynucleotide, (iii) thesequence of the polynucleotide, (iv) the secondary structure of thepolynucleotide and (v) whether or not the polynucleotide is modified. 7.A method according to claim 1, wherein the one or more characteristicsof the polynucleotide are measured by electrical measurement and/oroptical measurement.
 8. A method according to claim 7, wherein theelectrical measurement is a current measurement, an impedancemeasurement, a tunnelling measurement or a field effect transistor (FET)measurement.
 9. A method according to claim 1, wherein step b) furthercomprises contacting the polynucleotide provided in step a) with apolynucleotide binding protein such that the protein controls themovement of the at least one strand of the polynucleotide through thepore.
 10. A method according to claim 9, wherein the method comprises:b) contacting the polynucleotide provided in step a) with atransmembrane pore and a polynucleotide binding protein such that atleast one strand of the polynucleotide moves through the pore and theprotein controls the movement of the at least one strand of thepolynucleotide through the pore; and c) measuring the current passingthrough the pore as the at least one strand of the polynucleotide moveswith respect to the pore wherein the current is indicative of one ormore characteristics of the at least one strand of the polynucleotideand thereby characterising the double stranded target polynucleotide.11. A method according to claim 9, wherein the polynucleotide bindingprotein is derived from a helicase.
 12. A method according to claim 1,wherein the membrane is an amphiphilic layer or a solid state layer. 13.A method according to claim 1, wherein the transmembrane pore is atransmembrane protein pore.
 14. A method according to claim 13, whereinthe transmembrane protein pore is derived from Mycobacterium smegmatisporin (Msp), α-hemolysin (α-HL) or lysenin.
 15. (canceled)
 16. A methodfor modifying a target double stranded polynucleotide forcharacterisation using a transmembrane pore in a membrane, comprising(a) ligating a Y adaptor to one end of the polynucleotide and ligating ahairpin loop adaptor to the other end of the polynucleotide; and (b)attaching to the Y adaptor one or more first anchors for coupling thepolynucleotide to the membrane, attaching to the hairpin loop adaptorone or more second anchors for coupling the polynucleotide to themembrane and thereby providing a modified target double strandedpolynucleotide; wherein the strength of coupling of the hairpin loopadaptor to the membrane is greater than the strength of coupling of theY adaptor to the membrane.
 17. A method according to claim 16, wherein(a) the hairpin loop adaptor comprises more anchors than the Y adaptor,(b) the strength of coupling of one or more second anchors to themembrane is greater than the strength of coupling of the one or morefirst anchors to the membrane, (c) the strength of coupling of the oneor more second anchors to the hairpin loop adaptor is greater than thestrength of coupling of the one or more first anchors to the Y adaptor,(d) the one or more first anchors and one or more second anchors coupletheir respective adaptors to the membrane via hybridisation and thestrength of hybridisation is greater in the one or more second anchorsthan in the one or more first anchors, (e) the one or more first anchorscomprise one or more rigid linkers and the one or more second anchorscomprise one or more flexible linkers, or (f) any combination of (a) to(e). 18.-25. (canceled)
 26. A population of adaptors for modifying atarget polynucleotide for characterisation using a transmembrane pore ina membrane, wherein a proportion of the adaptors are Y adaptorscomprising one or more first anchors for coupling the polynucleotide tothe membrane, wherein a proportion of the adaptors are hairpin loopadaptors comprising one or more second anchors for coupling thepolynucleotide to the membrane and wherein the strength of coupling ofthe hairpin loop adaptors to the membrane is greater than the strengthof coupling of the Y adaptors to the membrane.
 27. A population ofadaptors according to claim 26, wherein each adaptor comprises a doublestranded MuA substrate. 28.-29. (canceled)
 30. A method according toclaim 10, wherein the polynucleotide binding protein is derived from ahelicase.
 31. A population of adaptors according to claim 26, whereinthe population of adaptors comprises a pair of adaptors.